السلام عليكم ورحمة الله وبركاته بسم الله الرحمن الرحيم والصلاة والسلام على سيدنا ونبينا محمد وعلى آله وصحبه وسلم يا ربنا لك الحمد كما ينبغي لجلال وجيك وعظيم سلطانك اللهم افتح علينا فتوح العارفين اللهم معلمنا ما ينفعنا وانفعنا بما علمتنا يا الرحم الراحمين اليوم ان شاء الله سوف نتحدث about a female companion of the messenger of Allah صلى الله عليه وسلم her name is Al-Khansa رضي الله عنها and a male companion سيدنا سلمان الفارسي رضي الله عنه so let's insha Allah start with Al-Khansa her her name her phone name is to mother بنت عمر بن الحارث بن الشريد so on the 9 on the 8th year of hijra there was a lady an honorable lady who came to be with سيدنا محمد صلى الله عليه وسلم she she was a beautiful lady she her name was very well known and she was the very eloquent lady she was a poet and she used to say a lot of poetry and she is the Muhammad صلى الله عليه وسلم liked her poetry actually Al-Khansa is the poet who laminated her brothers and they were called Sacher and Muawiyah when they passed away and she was very eloquent in in limitation she enumerated all the good qualities all the good characters of her brothers along with mourning them so I'm gonna say two lines of poetry she said when she was talking about her brother صخر عينية جودا ولا تجمدا لا تبكيان لصخر الندا لا تبكيان الجري الجميلة لا تبكيان الفتا السيدا so she was she was so fond of her brother and when when he died of course at that time she was not a Muslim at that time and she she mourned him and she mourned his brother for years and years and she used to cry a lot and people used to empathize with her and they didn't know how to comfort her how to calm her she loved her brothers she was her life before Islam there is nothing to prevent her from doing from exaggerating the sadness over the dead people so if we stop here for a second and think about it when Sayyidina Ibrahim the son of Sayyidina Muhammad صلى الله عليه وسلم passed away after putting him in the grave he teared so they asked him the companions asked Sayyidina Muhammad صلى الله عليه وسلم what is this why are you crying he said إن العين لتحزن وإن القلب لا يحزن وإن العين لتدمع ولا نقول إلا ما أمرنا الله إنا لله وإنا إليه راجعون indeed the eyes tear and the heart mourns the heart feels sadness feels the loss but we do it in a reasonable way we do it in an acceptable way and this سبحان الله this means that we we all lose loved ones everyone is gonna die no one is living for it forever but when we mourn our loved ones we have to do it in an Islamic way سيدنا محمد صلى الله عليه وسلم said you can have sorrow and sadness for three days morning is for three days except for the widow she stays for four months ten days in her atta سو this is this this way of exaggeration for for the death of her in the sadness for the death of her brothers was not accepted in Islam سيدنا محمد صلى الله عليه وسلم we just have to remember that this is Allah's decree death is going to strike everyone death is but a door someone opens that door and passed by before us we are all going through that door سو what eases us at the time of at this hard time is that we are all going to meet when we pass this door so this will make it a little soothing for the heart سيدنا محمد صلى الله عليه وسلم now what happened to Al-Khansa after she became a Muslim so Islam changed the life of Al-Khansa and there was a delegation of Bani Sulaym who came to سيدنا محمد صلى الله عليه وسلم she was with them سيدنا محمد صلى الله عليه وسلم and at that time immediately she erased all the ignorance qualities that she has سو before Islam before someone becomes a Muslim this is the ignorance time for him and when she became a Muslim she changed completely سو what is this ignorance time isn't just for people who are not Muslim and they became Muslim so that the time before becoming a Muslim is called this سو no each and every one of us has an ignorance time an ignorance period someone might not be practicing and then Allah سبحانه و تعالى guided him guided his heart and he became practicing سو he is a Muslim but that period is called the ignorance period loving dunya is from ignorance when we when we love Allah سبحانه و تعالى we realize how this should not be in our life as I mentioned earlier we want dunya we want to be rich but we want the dunya to be in our pockets not in our hearts سو she used to come to say in the Muhammad صلى الله عليه وسلم to attend the gatherings in the masjid and she used to listen to the Quran that say in the Muhammad صلى الله عليه وسلم used to recite سو when someone listens with his heart it's different than someone listening with his ears how سو sometimes same ayas are recited in in image lists the image lists people are gathering just a few ayas are recited you look at the faces you might find someone crying out of and someone might be just looking right and left not affected at all so when there is a gathering of remembering Allah سبحانه و تعالى try to be there with all your limbs with your heart with your eyes with your ears with everything so this is what happened to Al-Khansa she opened the doors of her heart to let the light of everything that say in the Muhammad صلى الله عليه وسلم is saying get into her heart so سو the first thing she did she started to change the way that she was raising her for children for boys سو she instilled in them the love of Allah سبحانه و تعالى she instilled in them the love of Sayyidina Muhammad صلى الله عليه وسلم she instilled in them the love of fighting for the sake of Allah سبحانه و تعالى of sacrificing themselves for the sake of Allah سبحانه و تعالى so Al-Khansa رضي الله عنها was a very good Muslim companion she witnessed conquering so many countries and during the time of عمر بن الخطاب رضي الله عن The Qadisia battle took place so سيدنا عمر sent the army to free Iraq and to spread Islam there and the battle that happened was was called Al-Qadisia سو she was talking to her four children for four young men and she was reminding them of how great is to have to be a murder for the sake of Allah سبحانه و تعالى and she reminded them that victory is from Allah سبحانه و تعالى so it will be either victory or to become a murder سو she said يا بنيا إنكم اسلمتم طائعين و هاجرتم مختارين so you became Muslims by your free will and you migrated with your own choice سو just remember إعلموا أن الدار الباقية خير من الدار الفانية just remember that the eternal life is way much better than the vanishing life so she is emphasizing to her children that the آخرة the day after the life after is much better than this دنيا they are not comparable and in this سيدنا علي رضي الله عنهم ساد إعمل لدنياك كأنك تعيش أبدا وعمل لآخرة كأنك تموت غدا so live for your دنيا and work for this دنيا then you think that you will be living forever and live for the آخرة work do your best for the آخرة it's as if you are dying tomorrow how do we understand this سفر the first part if you are living just for this دنيا then آه the human human being would would think would plan would do things would think of what will happen after 10 years what will happen to my children so the the the human being will think for this دنيا as if he is living for for for a long time forgetting that سيدنا محمد صلى الله عليه وسلم said كأنما استظلة تحت ظلي شجرة the one who is living in this دنيا is like the one who is traveling he got tired so he stayed and sheltered had some shelter under under a tree had the shade of the tree just for for a few minutes so this is our life the few minutes that we that this person had the rest this is life but the آخرة is eternal while when someone thinks of the آخرة he will remember حديث رسول الله when he said صلي صلاة كأنك مودهر pray your prayer perform your prayer as if it is the last prayer that you are going to pray as if that you are dying immediately after so living for دنيا or living for آخرة there is no comparison this is what الخنصة raised her children to so she said she said her words and she advised them and she gave them her words of wisdom and she said tomorrow if you wake up you will go to the battle and you should remember that if you die you will be in the high heavens with سيدنا محمد صلى الله عليه وسلم so this is what we are seeking to be reunited with سيدنا محمد صلى الله عليه وسلم so her her children woke up the next day they they were so courageous they wanted to go and fight for the sake of الله سبحانه وتعالى and during the battle all four of her children passed away they became martyrs now compare her reaction to the death of her of her children to the reaction of her of the death of her brothers so when when her children passed away when her children were killed in the in the battle she said الحمد لله الذي شرفني باستشهادهم الحمد لله she thanked Allah سبحانه وتعالى that he had honored her with four murders وأرجو من ربي أجمعني بهم في مستقر رحمته and I am begging Allah سبحانه وتعالى that he would reunite me with them with سيدنا محمد صلى الله عليه وسلم so she did not do what she did with her with her with her brothers you see how Islam changes the personality so when we practice when we practice Islam as it is as سيدنا محمد صلى الله عليه وسلم then we are guided then we are protected by Allah سبحانه وتعالى when we have anything to strike us when we have any calamities when we have any hardship كن مع الله في الرخاء يكون معك في الشد be with Allah سبحانه وتعالى at the time of ease he will be with you at the time of hardship so when we understand the reality of things we act differently and we ask Allah سبحانه وتعالى always to guide us always to help us always to be with us and when we depend on him we know that we will be saved in this dunya and in the day after insha'Allah so this was the story of Al-Khansa now we move to سيدنا سلمان الفارسي الفارسي is the Persian so سلمان the Persian the story of سيدنا سلمان is amazing he is an amazing person سبحان الله سلمان رضي الله عن كانت ممتازا جدا سيدنا سلمان was very rich so he was a person who lived in Asfahan and he was the governor the chief of the city of the village that he lived in so سلمان says I was loved the most of people to the heart of my father he was so loving he was so caring to the degree that he put me he restricted me of going out of the house because he is afraid that something might happen to me so they were worshippers of fire and because of his extreme love to his son he made سلمان قطنو نار and the word قطنو نار means the servant of fire so he would lit the fire he would not let the fire go down he would always ignite it he would always add the wood he is the one who takes care of the fire and this is a very important position for the people who worship fire so his father as I mentioned was very rich and he on the village one day he said I want to go out so the father accepted and he سلمان was walking and he passed by a church so when he passed by a church he heard some noises inside the voices of the people who were praying so he said to himself I have never liked worshipping fire I have never liked this I have never accepted this I feel there is something wrong in this he feels that the faith of his father is and the people around him is corrupt so he hated it from deep inside so when he heard the people praying in the church he said I think my father put me inside the house and prevented me from going out so that I will not meet a people on another religion they thought that this was their good religion so when he entered the church he looked at them and he liked what they were doing so he said oh this is much better than the religion my people worship and he stayed with them until the sun set so when he came back home his father said to him where are you? when I was at the church so he said my father I passed by people praying in the church and I liked what they were doing I liked their religion and I had been with them all day until sunset and the father said oh boy there is no no hair in this religion you have to be on the religion of your fathers so he chained his son he chained Salman at home and he would not allow him to go away but before leaving the church Salman told them so where is the religion the origin of this religion what is the origin of this religion so they said it's in Sham so he came back and this was what happened his father chained him he did not want them want him to go anywhere but he left a message for them that if anyone is going there if any merchants are going let me know I want to go with them and subhan Allah a group was going so they sent him a message or someone to tell him that the group is going so he ran away and he went with them so when he arrived in Sham he said who is the most religious person here and they showed him the bishop of the church so when he went to him he told him that I love this religion and I want to be with you to serve you in this church so I would learn from you the bishop told him yes come in but that person was very bad he was ordering people to give charities but he would keep this charity for himself he would not give the poor so Salman hated him and that bishop after some time he passed away so they assigned another person to take his place but that person was way much better than that bishop so he loved him and he stayed with him a long time so until it was his death so Salman asked him who do you want me to go to who do you want me to follow I don't know who is good who is bad and he said there is a person in الموصل in العراق so you go to him he is on the right path so when he died and he was buried so he went and traveled to الموصل to meet that person and he also found him a good person on the right path and again in the same time he went to him so he asked him how and where would I go and he told him there is one person in نصيبين and he is on the right path so Salman traveled and went to نصيبين and he also found him a good person until it was his death time so he asked him where to go who is to trust and he told him there is a person in نعموريا and that is the Roman's land so he went to there and he stayed with him he was also a good man but on his death bed he asked him tell me what to do where to go because he liked the religion he didn't want to go back to where to go because he liked the religion he didn't want to go back to to worshiping the fire which he hated a long time ago so that person told him it is now the time of a new prophet and this new prophet is on the religion of إبراهيم عليه السلام on the same way that إبراهيم عليه السلام so you will find him in a place where there is palm trees and he has some characteristics some qualities that you will not miss he accepts gifts he does not accept and there is the seal of prophecy on his shoulders so if you can't follow this person you will be saved he traveled to go to that place but on the way the person who who was traveling with him he got his money and his few cows and he sold him to a person who lives in Medina so as soon as he arrived in Medina he loved the place because it's the place where there are palm trees so he said I hope this is the place of the new prophet so he was working until one day the cousin of the master came and he was talking to him and he said they were talking to each other the cousins to each other so he said so he was cursing the people of and because they are gathered about they are gathered around a person who came from and he he claims that he is a prophet at that time Salman was up the palm tree getting some dates so he was about to fall down from the palm tree so he went down immediately and he said what did you say so his master looked at him and he hit him and he said why are you asking you have nothing to do with this why are you asking so he said nothing nothing I just heard something and now your cousin just confirmed it so he this is what he answered him and during the night he collected his stuff and he went to so he he was so eager to see him he wanted to to make sure that he is the person so he came to and he said to him إنه بلغني أنك رجلون صالح I heard that you are a righteous person ومعك أصحاب لك غرباء ذو حاجة and I know that you have migrated and you have people who are poor and I have this this thing for صدقة for charity so I would give it to you and not to anybody else so he offered it to say المحمد صلى الله عليه وسلم and to the companions so سيدنا محمد صلى الله عليه وسلم said to the companions take it eat from it but سيدنا محمد صلى الله عليه وسلم did not eat from whatever سلمان gave them so he said to himself well this is the first one now then he said to سيدنا محمد صلى الله عليه وسلم I collected my stuff and I noticed that you do not accept صدقة you do not eat from the صدقة but these things this food is a gift to you so سيدنا محمد صلى الله عليه وسلم ate from it and he gave some to his companions also so again he was so happy and he said this is the second one it came true then he came once to سيدنا محمد صلى الله عليه وسلم and he was in وقيع الغرق following أجناز and his companions were around him so سلمان wanted to see what is in between the shoulders of سيدنا محمد صلى الله عليه وسلم so he went behind him سيدنا محمد صلى الله عليه وسلم felt that and he put down his gown he made his the seal of prophecy shown and clear for سلمان so when سلمان رضي الله عنه so that he said فانك ببتوا عليه وقبلوا هو أبكي I hugged him and I kissed him and I was crying and crying so سيدنا محمد صلى الله عليه وسلم said come come in front of me sit in front of me so he سلمان told his the story the whole journey that he went through until he met سيدنا محمد صلى الله عليه وسلم and until he accepted Islam and he was sitting with سيدنا محمد صلى الله عليه وسلم now الله سبحانه وتعالى has guided سلمان رضي الله عنه so his his ending up in المدين المنورة was not a coincidence after all this long journey from one place to another place from one travel to another travel he was looking for the truth he was looking for someone to tell him about Islam he was looking for سيدنا محمد صلى الله عليه وسلم just to say to him أشهد الله إله إلا الله وأشهد أنك محمد الرسول الله سلمان رضي الله عنه when he became a Muslim he impressed سيدنا محمد صلى الله عليه وسلم with the great knowledge he had he traveled a lot so he saw a lot and he had a lot of experience and also he read a lot of books for Persians Romans Jewish and he was known to be very smart wise and a very good person so in مدينة سيدنا محمد صلى الله عليه وسلم had the brothership between the companions and سيدنا محمد سيدنا سلمان was the brother of سيدنا أبو الدرداء مشاء الله so both people are people of knowledge سيدنا محمد صلى الله عليه وسلم said about أبو الدرداء هو حكيم حكيم أمة هو أبو الدرداء من أمي و عن سيدنا سلمان he said لقد أشبع سلمان و علمان سلمان has a lot of knowledge مشاء الله so this is the شهادة this is the way that سيدنا محمد صلى الله عليه وسلم used to look at both of them now سيدنا محمد صلى الله عليه وسلم is in مدينة he built the masjid in Medina and then after settling and getting stronger there were the battles started so there was the battle of بدر then the battle of أحد during both سلمان could not participate because he is still a slave you know that he was sold he was sold to someone and that person still owns him so one day he came to سيدنا محمد صلى الله عليه وسلم and سيدنا محمد صلى الله عليه وسلم told him كاتب يا سلمان he asked him to talk to his master and ask him how much does he need to pay for him so to set him free and سلمان سلمان said I don't have anything he said don't worry about it so he asked his master what do you need so to free me and that person said ثلاث مئة نخلة واربعين أوقية 300 palm trees وحيها له بالفقر he wanted small palm trees so that he would grow them for him for him and then they will grow later so he would plant them for him and they will grow later so he wanted 300 palm trees and 40 أوقية each 5 5 أوقية is 1 kg which is 2 lbs so about 16 16 measure to to get him free so what is the measure in gold سبحان الله سبحان الله so سيدنا محمد صلى الله عليه وسلم said he said to to the companions help your brother help your brother so someone would come to سلمان and give him 30 sprouts of palm trees small palm trees and the other would come with 20 someone would come with 15 someone would come with 10 until he got all the 300 300 sprouts of the palm trees so سيدنا محمد صلى الله عليه وسلم go prepare the land dig the holes but do not put anyone in the holes when you are done digging 300 holes I will come myself so سلمان رضي الله did and he help his companions his companions helped him and until everything was ready so he called سيدنا محمد صلى الله عليه وسلم who came and he planted all 300 palm trees and until today these 300 are the عجوة of مدينة so when we go to مدينة and we buy this small black dates this is the عجوة of مدينة that's a سيدنا محمد صلى الله عليه وسلم planted himself so now he got the palm trees without the the gold so سيدنا محمد صلى الله عليه وسلم was once given an egg of gold a small egg of gold someone gave it to him so he called سلمان رضي الله and he told him take this and pay your ransom of the money and he did and سيدنا سلمان رضي الله عن was فريد he is not a slave anymore a short time passed and there was the battle of the trench and it was the first battle that سيدنا سلمان would participate with سيدنا محمد صلى الله عليه وسلم and that was during the fifth year of the hijrah سبحان الله so the Muslims سيدنا محمد صلى الله عليه وسلم got the information that قريش united with غطفان and other tribes and they wanted to come and kill the Muslims and to destroy them they were about 10,000 soldiers the non-Muslims so سيدنا محمد صلى الله عليه وسلم said he talked to all his companions and he told them what's going on so سيدنا سلمان said to رسول الله صلى الله عليه وسلم يا رسول الله إنا كنا بفارس إذا حصرنا خندقنا علينا نحن في فروجيا إذا كنا كنا حسن نحن جمعنا نحن so this is what what we used to do and سيدنا محمد صلى الله عليه وسلم liked that idea and he thought that this is an amazing plan so he ordered يجب عليهم أن تبدأ تقريباً، لذلك لديهم ترنج من نور مدينة because there were all gardens and orchards and the other side so that was the only place that they would attack them from. لذلك لديهم all working, سيد محمد صلى الله عليه وسلم divided the amount of digging how much each person would get, he grouped the Muslims into several groups and each group had to dig some a few meters down and around the place. سيد محمد صلى الله عليه وسلم used to say used to make dua for the Muslims for the companions and he would say اللهم إن العيش عيش الآخرة فغفر للأنصار والمهاجرة they oh Allah the real life is the life after ya Allah I ask you for forgiveness for those for الانصار who helped those who migrated from Makkah so the people of Medina and the people of Makkah who came with سيد محمد صلى الله عليه وسلم who migrated to Medina. سيد محمد صلى الله عليه وسلم so they were eager to work harder when they heard the dua of سيد محمد صلى الله عليه وسلم and they were working so hard with with happiness and they would answer نحن الذين بايعوا محمدا على الجهادي ما بقينا أبدا we are the one who gave glids to سيد محمد صلى الله عليه وسلم that we will fight fight with him as long as we live. سلمان was a very strong man he worked what equals 10 of other companions he was very strong he was a very well built man so المهاجرين the people of Medina would say سلمان مننا سلمان is from us and the people of Makkah they would say no سلمان is from us so سيد محمد صلى الله عليه وسلم to listen to them and he heard what they were arguing about so he gave the last word and he said سلمان مننا على البيت سلمان is from us the people of the house so he was given the honor to be from the family of سيد محمد صلى الله عليه وسلم and they were working hard then there was a big big rock that could not be removed by سلمان and to say the Muhammad صلى الله عليه وسلم so how hard it was for him to do to dig it so he came he took the shovel from the from the سلمان and he hit the the rock and there was a flash of light then he hit it another time there was another flash of light then he hit it a third a third time and there was a another flash of light so سيد محمد صلى الله عليه وسلم so سيد محمد صلى الله عليه وسلم said the first hit there was a flash there was light that Allah سبحانه وتعالى showed me that Yemen is to be conquered by Muslims the second one is Sham and Morocco will be conquered by Muslim the third one the far east countries will be conquered by Muslim which means that Islam will spread all over east west so this was how سيدنا سلمان worked with the Muhammad صلى الله عليه وسلم in the trench and when when when the whole work was done in six days and when they when the non Muslims arrived into the place and they found the trench they said this is something that the Arabs would not know we don't know this and they were told that there is a person person amongst the Muslims and he told them what to do so for سلمان was not just a person person سلمان was a person of the house of سيد محمد صلى الله عليه وسلم now the battle ended the trench was one of the big factors of the victory of the Muslims and سيدنا سلمان thank Allah سبحانه وتعالى for that he thanked Allah سبحانه وتعالى that he gave him the courage to say the idea to say the Muhammad صلى الله عليه وسلم and there is a big important lesson here when you do something that it becomes so successful so good so so many benefits out of it then thank Allah سبحانه وتعالى that he provided you with the means to do so it's the blessings that Allah سبحانه وتعالى has given to you so that you are successful so when you think Allah سبحانه وتعالى you will increase you سبحانه وتعالى and there are so many forms of thanking Allah سبحانه وتعالى and inshallah one day we will talk about these forms and الله سبحانه وتعالى loves to hear the voice of his righteous people so when we say الحمد لله الله likes to hear that so he would increase ولا ان شكرتم لأزيدناكم he said it he promised in the Quran if you thank me then I will increase you so this is what happened there are also some other points about سيدنا سلمان but I think we are a little bit over time so inshallah I would like to end with one thing that when سلمان رضي الله عنه was sick on his deathbed he cried and the companions were around him and they asked him why are you crying he said اما والله ما ابكي جزعا من الموت I'm not afraid of death ولا حرصا على الرجع I don't want to live anymore ولكن انما ابكي لأمر عاهده إلينا رسول الله صلى الله عليه وسلم أخشا أن لا نكون حفظنا وسية نبينا but I'm crying that سيدنا محمد صلى الله عليه وسلم asked us for something ordered us to do something but I'm afraid that we lost this thing we did not fulfill this promise so he said to us ليكم بلاغو أحدكم من الدنيا كذادي الراكب so let the what what a person owns in this دنيا be what someone who is traveling can hold with him لذا let's think for a minute now if we open our door ops and we looked at how many clothes we have how many shoes we have how many cars we have how many suits we have لنفكر again and try to to to donate what we don't want to try to give people and remember that when you give الله يوحبه أطيب don't give the the torn one or the راث one just give something good for the sake of Allah and remember that he will save it for you in the day after سيدنا سلمان looked at his wife and he said what have you done with the mist that we brought with the perfume that we brought so she said here it is so he said put it in water and spray it spray it around me فإن الآن يأتينا قوم ليس بإنسن ولا جن now I am being visited by some some people some someone by some spirits who are not humans who are not سو another thing that we need to pay attention when we have someone when we we know that someone is dying we need to to make out in the in the room because the angels are coming we wanted to be pleasant for them and also one important thing to do is try to get a few drops of water on the lips of the person who is dying because at that moment الشيطان will have a drink of water and he will say just say just don't believe in Allah and I will give you this water to drink the man the person who is dying would be thirsty and that's why they put drops of water in his in his mouth سو this is why we make the دعاء اللهم إنا نهو ذو بك من فتنة المحيا والممات فتنة الممات is the we seek refugee in New York Allah from the fitنة of death this is what شيطان wants even at the very last breath of our life he wants he will not get this bird he wants to get to the people away from the right path he want to get to the people to be non believers even when they are they are dying سو this was the story of سيدنا سلمان رضي الله عنه so just a very interesting very amazing story there is nothing in coincidence when Allah سبحانه وتعالى wants you to do something then it will happen and it's all predestined وصل الله على سيدنا محمد وعلى آله وصحبه وسلم يا ربنا لك الحمد كما ينبغي لجلال وجيك وعظيم سلطانك وسلام عليكم ورحمة الله وبركاته


Now, let's talk about AI when we look at AI. You folks have been leveraging AI for a long time, but now we have started talking about Generative AI, Gen AI. Gen GPT is a big buzzword these days.  I really do appreciate the fact that when I got the transpose that we had an AI strategy already. And so what I like is that when others were announcing, I don't know, we have AI capability, we already had it. And so we've done a couple of things. Throughout the incident process, if you are running an incident in transposite, you know, we're friendly with Slack, we're friendly with transposite UI. So it doesn't matter where the communication is taking place. One of the things I truly appreciate about transposite is as you're going through that incident, as you're having those conversations, you're not having to dictate to a scribe or take time from the resolution process to scribe. We actually have an AI teammate that is listening, watching that conversation and providing that context. We even prompt for you, say, man, look, this appears to be an update to this incident. Something has changed. Would you like to change that? We provide that functionality out of the box. And then at the end of the incident, what we hear is a lot of people like to run a postmortem. You'll never hear me say postmortem. That's what our customers say. I call it a post incident review. I do that because there's a lot of learnings to be had there. I cannot tell you the number of times I've seen someone go through that timeline and pull the important pieces out. It's error prone. It can easily misunderstood as to what action was taken by who at what time. And so I think the benefit of our AI is that summarization includes key events that took place during that incident. This person did this. This person mentioned this. This decision was made by the group. And we're doing that throughout the entire incident life cycle. And so by the time you finish your incident, we're gonna give you a pretty good head start on your post incident review. I think that's a big one. But we're not gonna stop there. I mean, like now that it's out there and now that we see a lot of this, ideally what good is a hundred integrations if I cannot suggest that you use one? If you're taking this action every time you have an incident, we wanna enable you to say, do you wanna do this from now on?  So getting away just from, not just from the summarization aspect, we wanna be a suggestion recommendation engine and that is huge. Like being a recommendation engine or a suggestion engine based on someone's ongoing conversation and the tools that they're already using is a superpower and one that we're uniquely designed to solve for.


So far, we've learned about RNA-seq, some background, learned about library construction, how to generate the sequences. Now we get to the important part where we take the sequences and we do some alignments and visualization. So in this module, the focus is to look at some of the challenges that we face when we deal with RNA-seq alignments. What are some of the alignment strategies that are available out there? We will be focusing on bowtie and top hat and go over how these tools work. And then after that, we'll go over the output files from the alignments, so the BAM and the bed formats. For those of you who are not familiar with those formats, we'll go through the formats what they look like and how you can manipulate them and what you can do with them to do further downstream analysis. And the final part will be alignment QC. So we have talked about QC at various points in this pipeline. So we've talked about QC at the library construction level, when we talked about RINs. We also talked about QC at the FASQ level where you're assessing the sequences themselves to see how they were sequenced. But here, we're going to be looking at post-alignment. So after you align, after you figure out where these sequences are coming from in the genome, we can do a lot more QC assessment at that level. So I'll give you some examples of some QC metrics that you can look at to assess the quality of your sample and to give you more information and some biological information about the sample that you're looking at. Now, when it comes to RNA-seq alignment, we face a few challenges. So some of these challenges include the computational cost. This topic was touched upon multiple times today. But each lane of sequencing, again, depending on what kind of sequencer you use, but if you're using, for example, high-seq 2,500, a lane of sequencing can generate 300, 400 million reads. And usually, you do one to two samples per lane. So we're talking about hundreds and millions of reads that you're going to have to take and align to the genome. So that process is actually computationally expensive because you will need, just like we're doing today, you're going to need some computational instances, such as Amazon. And the process is also time-consuming. And not only that, but the amount of data you generate, there's a lot of data. You get alignments, you get fusions. So you have a lot of files that you have to store and the footprint is a lot more here compared to other data types, such as DNA. When it comes to alignment as well, we get to deal with introns. So these are large gaps that the aligner has to recognize. These are things that aligners in DNA sequencing, they don't have to deal with these large intronic gaps when you try to map them back to the whole genome, but they have to consider when you're dealing with RNA-seq data. Also, sometimes you ask, if I run it once, will I be done? No, there are a lot of updates that happen to the reference, a lot of updates that happen to the gene annotation. There are a lot of tools that come out that will look at different aspects of the RNA-seq data. So chances are you will go back and reprocess your data multiple times depending on how many updates happen and how long your project is going for. Now, when it comes to the RNA-seq mapping tools, you can classify them into three different categories. So there is the de novo assembly tools, you have the tools where you align to transcriptome reference, and tools that you align to a whole genome reference. So what strategy is best? Now that depends on what data you have and what you're trying to achieve. So for example, if you do not have a reference or your reference is not sequenced, then you tend to do de novo assembly. So there are tools that will do that for you. You also tend to do de novo assembly. There's a lot of complexity or polymorphism in your data set and you don't wanna force it into a reference. So we talked about variations, we talked about how the reference is only representing certain genomes and you don't wanna force your variations into that one genome. You can go ahead and do de novo assembly because that will deal with the complex polymorphisms. You can also do, there are tools that will align to the transcriptome. So these are references that will have all the possible combinations of transcripts and isoforms in the genome and you're simply just taking your mRNA or CDNA and you're trying to map it back to the transcriptome without having to deal with assembling the transcript or isoforms. But the most popular type of aligners are the ones where you get to take the CDNA and try to align it back to the whole genome reference and do some sort of an assembly of the isoforms. And again, each one of these strategies, it comes with a complex and a set of tools and packages that you can try. Now, in terms of tool, here is just a timeline of all the different aligners that are available out there and when they were developed. And the ones that are for RNA seek their highlight in red and this is 2015, so it's pretty recent. And you can see that there are so many different tools that are being developed every year, different aligners to be more specific. And for this workshop, we have decided to pick two tools. So we're doing top hat and I don't know if we have time to do star. The commands are available. Now top hat is a bit more established. It's been in the community for a while and we like to start with top hat because of the fact that it's very established and it's user friendly. There's a big community out there. So the likelihood that if you run into a problem, if you have a question, most likely that it's actually been answered, someone else has faced that problem. And yeah, so that's top hat star is another great tool that has been recently developed. It's a lot faster. That's one of the biggest advantages of using star as an aligner. It's a lot faster than top hat. But as I said, we'll get to use both. Again, there are a lot of other aligners. However, once you learn to use one tool, you can take those goals and just apply them to any other tool out there. Just a matter of learning the specific parameters that come with every tool. Should I use splice aware or unsplice snapper? Again, so it's going back. We know that the RNA that we have does not contain entronic regions, but if you plan to align it to a whole genome reference, then the whole genome reference is DNA. It contains entron and exon. So you will have to deal with or pick a tool that is splice aware. If you're aligning to transcriptomic reference, then you don't have to pick a tool that is splice aware. Now, Bowtie and top hat, the package is splice aware aligner and splice junction detector. So the way it works is Bowtie is actually the backbone aligner for this package. Bowtie is great at aligning short reads. However, the thing that Bowtie cannot do is it cannot deal with very large gaps. Those are the entronic gaps. And that's why top hat works like a wrapper. It uses Bowtie as an aligner and it takes all that information from the alignment to piece the information together and create the splice junctions. And I'll go through an example of how it does it one step at a time. So let's take this as an example. Let's assume that we have two reads, read X and read Y. And this right here is the reference. So in this reference, we have an exonic region, entronic region and another exonic region, exon one, entron, exon two. And we're gonna assume that the reads that we're dealing with, they are long enough that they could span multiple exons. So read X in this example spans two exons, exon one and exon two. But read X, again, as a reminder, it's a CDNA coming from mRNA, so there is no entronic region in that piece. Read Y, we can assume that it's a read that spans one exon. So the way it works, top hats, well Bowtie is gonna try first and align all the reads to the whole genome. And anything that perfectly maps, then it will put it in an aligned bin or perfectly aligned bin. And anything that partially maps or partially un-maps, depends where you look at it, and then it's gonna put it in an aligned bin because those reads, there is a potential that they span multiple exons. So top hat will deal with those reads later. So top hat will take all the unaligned bins from Bowtie and it will try to split the reads into smaller segments. So it will take read X and split it, in this case into three segments. And you get to decide how many segments you want because you get to decide the length of each one of these segments. By default, it's 25, I believe. I don't know if that has changed. And then you take each one of these segments and then Bowtie will align, will do alignment again. So align it to the whole genome again. And you will notice now, previously the read did not map, but when you break it down, the first piece will map the exon one. The second piece will still un-map because it's an exon-exon boundary. And then the third piece will map. So top hat will collect that information from Bowtie. And it will try to estimate a site where the splice junction is actually happening based on the mapping information of the small segments to the whole genome. And it will do that for all the reads that did not map initially. And it will build some sort of an index of all the possible sites of splice junctions. And once it comes up with that list, it will take all the reads and align them again to the sites that it created. To the Slice Library. And while it's doing that, it also looks at the coverage distribution across the different pieces and to make sure that it matches and so on. So is that process clear? The alignment process and the splice section? Questions? Okay. What happens to X2 in this one? So the reason why it's doing that is it's trying to figure out where the splice junction is happening. So because X1 aligned and X2 did not align but X3 aligned, it knows that the splice junction is between X1 and X3. So it tries to estimate based on the mapping and unmapping information of these little segments. Well, you're using it to obtain the splice sites. Once you get the splice sites or the predicted splice sites, you go back and align all the reads, the full read, not the chunks, to those splice sites to see if it actually is aligning properly and if the splice sites that they detected are proper. Yes. In case for the X1 matches with multiple and matching with X1, five? Yeah, I'm not sure how it deals with... I'm sure there is a filter for that, for pieces that map to multiple locations. Also, there is a different way of assessing mismatches, I believe, depending on the location within the read. If it's at the beginning of the read, then it allows a certain number of mismatches, but if it's at the end of the read, then it allows more just because the end of the read is known to have prone to errors, technical errors more than the beginning of the read. So I'm sure it deals with that aspect of multi-mapping. And you wanna, when you're deciding the length, and that's something we'll do in the integrated assignment, when you're deciding the length of those segments, you don't want them to be extremely short that they get to a point where they're not unique anymore and they map to so many different places in the genome because that will introduce a lot of false splice junctions in your data. Okay. Does the cycle once? So it does the cycle once, yes. So it does that for when we will run the alignment, you will see that it does it for the read one and read two, and it does that once. And we'll look at the output while we're aligning, and I think the process will make more sense as you follow the log while it's doing the alignment. Okay. Now, should I allow multi-mapping in top hat or in RNA-seq? Now, it really depends on the application. When you're dealing with DNA-seq, for example, oh, sorry, question. Yeah, I'm thinking about the previous slide. Yeah. How was you mean that it doesn't have something that binds to one side, but it's a fusion with this? So for fusion, it's a different algorithm. So this will say, this happens to be a fusion rather than splicing this algorithm, right? You have to specify, if you're looking for fusion, then the parameters that it uses, the thresholds for the expected distance changes. So here it's expecting, based on your library size, because you tell it the library size or it tries to predict your library size. So it has an expected distance between two breakpoints. But if you turn the fusion on, then the expected distance, the threshold will be increased and then it will consider it to be a fusion. Sorry, so yeah, multi-mapping. When you're dealing with DNA-seq, that's a common thing that you will encounter where a read can map to multiple places. And then depending on the tool you use and depending on what you wanna do, sometimes you can report all the reads that map to multiple places. And the tool can pick either the randomly pick the best alignment or pick the one with the highest quality and sometimes there are multiple reads that map with the highest quality so you can randomly pick one. With RNA-seq, it really depends on what you are planning on doing. If you're planning on doing varying calling, then I would say perform the same thing as DNA and try to not allow multi-mapping. If you're doing expression, you don't want to influence the dynamic range of your expression. So it's recommended that maybe you keep the multi-mapping so that the reads can map to multiple places. And same with Fusion, it's recommended that you would keep the multi-mapping. And that's an option that you will find in Top Hat. When we run the commands, you'll see there's an option that you can turn on and off.  You get SAM file, SAM stands for the sequence alignment map format. A BAM file is simply the SAM file. A SAM file is just a text file and the BAM file is the compressed version of a SAM file. And it's recommended that you keep the BAM version just because you will be saving space and you're not losing any information when you go from SAM to BAM. So it's safe to just keep the BAM and get rid of the SAM if you can. Now, there are a lot of tools that you can use to convert SAM to BAM, BAM to SAM, and vice versa. One of the ones that I use all the time is SAM tools. If you've heard of that, I use that a lot to convert back and forth. When you try to view a BAM file, you can't just simply open the file because it's binary. So you're gonna have to use a tool that will convert it to SAM, or just like the ZCAT version, you can stream the reads and look at them. However, that's not recommended because you will get millions and millions of reads so you can't really browse through the reads. Yes? How do the aligners handle SNPs? So as I said, so it allows for mismatches. It does. It does, and the rate of mismatches differ depending on where in the read it is. So at the beginning of the read, the rate is a bit lower than at the end of the read just because of the, I'll show you when you look at the QC, the quality of the bases in the read drops towards the end. So it allows for that. It's flexible that way. And you can change the number of mismatches if you want. If you wanna be more strict, then you can change that as well when you're doing the alignment. The default settings tend to be fairly permissible. So if you haven't seen a SAM or a BAM file, this is what it looks like. You can break it down into two sections. So you have the header at the top and you have the body. The header contains information about the sequencer. I'll get into the details of what the header actually includes in a bit. But the body contains the actual sequences that you have aligned. So all these sequences are the same sequences that you found in the FASQ file. And then it's adding another layer of information. It's that sequence in the FASQ file, but I'm telling you where it actually aligned and the quality of the alignment as well. So you can pretty much take the BAM file as well and then convert it to FASQ because all the sequences should be there, aligned and unaligned. I've talked about that. And BAM files actually, you can actually index the BAM files as well and that will make the retrieval of sequences a lot faster if you wanna pull a specific read coming from a specific position. You will have to index the BAM file to be able to pull those reads pretty quickly. So this is the header section I was talking about. What kind of information does it contain? So the tags that you add to HD, for example, it looks at the version of the BAM file. It looks at the sorting order. So how is this BAM file sorted? Is it sorted according to position or sorted according to the reads within the BAM? It also looks at the reference that was used to perform the alignment. What version of a reference was used? The contigs and their length and the species as well. Read group, so the library identifier. So it's all information that will help you. If you read the header, you should be able to go back and reprocess the data based on that information. So it'll tell you the library, it'll tell you the sequencing center, what type of sequencer was used and the sample name. And finally, the program that was used to perform the alignment and sometimes you get even like a command. The actual command that was used to get the alignment. And the richer the header is, the easier it will be for someone to reprocess the data without asking you questions or asking the person who sent the BAM file any questions. Now, the body of the BAM file contains a list of information. So I'm not sure if you can see the bottom. So I'm just gonna go through an example of tags that you can find in the alignment. So for each one of these sequences that are aligned, you'll get information. It's just the Q name. So that's the template name. It contains the read name and it depends to it the sequencing information. And it tells you whether it's read one or read two. You have the flag. So the ones that are highlighted will get into, I'll explain them in details in a bit, the flag and the cigar. These are the two that I'll explain in a bit. Our name, so it tells you the chromosome that the reads aligned to, the position within that chromosome, also the mapping quality and it will look at the pair, the read two. It will tell you where, what chromosome read two aligned to and what position of read two and how far is read two from read one. So that information can help you come up with an insert size distribution. So you can find the distribution of the size of the fragments that the libraries came from. And then you get the actual sequence and then another string for the quality. So for each base, you'll get the quality score, just like was discussed before with the FASQ files. So what's this flag that I've shown you earlier? So this is a 12 bitwise flag. It helps you, it describes the alignment. So they're trying to pack as much information about the alignment in this score. And this score is, you can think of it as a combination. It's a number from 1,000 to 2048, but it is represented in a hexadecimal representation. So it makes it a bit confusing. But each number represents a type of quality for that alignment. So it will tell you if the template has multiple segments or multiple alignments. If it will tell you if the segment actually mapped or it didn't map, if it was a proper pair, if the second read actually mapped as well in the same chromosome, it will classify it as a proper pair. If it's the first read or the second read, if it's a primary alignment or a secondary alignment, and if it passes filters, if it's a PCR artifact or if it's a supplementary alignment. So you can use these flags. If you use SAM tools, for example, you can use SAM tools and use that flag to pull all the reads that are coming from proper pairs. So you just provide, you don't need to provide anything, you just provide that flag that's associated with the description. And it will pull all the reads from that BAM file that are coming from proper pairs. It will pull all the reads that are coming from the first read, not the second read. So it just makes the retrieval of the reads from BAM file a lot easier. The cigar string is a string that's also in the BAM file. And that string is, so each read gets a cigar string and the cigar string explains the, it describes the actual alignment, the breakdown of the alignment. So if we take a look at this as an example, so saying 81M, 859N, 19M. So what does that mean? This means that there are 81 bases in that read that matched, then there were 859 gaps or bases that were gaps and then 19 bases matched. So the hundred bases read, the first chunk of it matched an exon and there was a big, a large intronic region and then another exonic region. So by reading this cigar string, it will help you visualize the read and where it aligned. And that's what actually BAM viewers or alignment viewers use, they pull the cigar string and then they use that information to help you visualize the read and what the breakdown of the read is. So you had a question. Yeah, what's secondary alignment? Secondary alignment, as I mentioned, if you have multiple, if the read aligns to multiple places and the primary alignment would be the best alignment and then if there are other locations for that alignment that are less quality, then that would be considered the secondary alignment. Yes? The flag, on your example, it says 99, how do I interpret that? Sorry? Yeah, so on the previous slide, on the example, it says 99, you see that 99. Oh, this one right here? Yeah, four flags, but then on your next slide, there is no 99. Oh, so it could be a combination of, so not one, so 99 is probably a combination of different qualities. So there is a website that I usually like to show. So let's try and look for it, where you can put the number and it will break it down for you and it will tell you what that mean, what combination of metrics that actually represents. Because it could be like a first read and it could be properly paired and it could be past quality and they combined it is a score. Let's see if we can find cigar, because it's kind of impossible for, oh, there you go. So this website is provided by them, so you put the flag, in this case it's 99, and it will tell you what combination of metrics it's actually coming from. So all I need to do, you don't have to put the whole link, all I did is I searched cigar flags explained and this is by Picard. So you can go here and if you're interested in a combination of filters, you can check all of them. So you can do the opposite, you can check the filters that you're interested in and the numbers will change. Then you take that number and you go to Sam Tools view, you specify the flag that you're interested in and it will subset the file. And when you're sub-setting the file, you can either include, make an inclusive list or exclusive list. So you can say I only want reads that fit these criterias or I want all the reads that don't fit this criteria and so on. So there's so many different ways you can subset and ban file. Go back. So we talked about the cigar string. The bet file is another format that is important when you're trying to subset a ban file. So a bet file is a text file that contains a few columns. It has the chromosome, start, end and whatever feature you're interested in. So let's think of, let's use an example. So let's say you wanted to pull a specific gene from your ban file. So in the bed you can say the gene has this chromosome, has this start, this end and this is the gene name. So you can use the bet file as a way to intersect the bed with the ban. So there are tools that will take the bet file that has the gene information, gene boundaries and the ban file that has the alignment and you tell it, okay, take these boundaries and overlap them with the alignment and pull the alignments that overlap this specific gene, just these boundaries. And it doesn't have to be a gene, it could be anything you want, a region in the genome, a band, whatever you want, as long as it has chromosome, start, end and a name, that's a bet file and you can use it to intersect with a ban file. And you can actually intersect two bed files as well if you want, it doesn't have to be a bet and a ban. So this is just a list of some of the tools that you can use to manipulate the ban file or find intersection with the bed. So SAM tools, as I've mentioned, I use that a lot. You can use BAM tools, you can use Picard. For bed files, you can use bed tools or bed ops. Now, for me, I mainly use SAM tools and bed tools. Bed tools is very, very useful, especially when you're looking at coverages, for example, because you can do that, if you wanna pull the raw number of reads that cover a specific region, you can use bed tools. How should you sort your alignments? There are two ways that you can sort your alignments, you can do it either at the biposition and this is mainly for performance reasons, so that the whatever tool that you're using downstream will have an easier time to retrieve the reads if it's looking for them biposition. You can also sort the ban file by read name and the intention behind doing it that way, sometimes, for example, if you're looking for fusions, you want to find out if the first read is mapping to a chromosome and the second read is mapping to a different chromosome and you want the reads to be right next to each other, so when you're doing the check, you can just check quickly. And there are other tools that you can use also for sorting the ban file. Now, IGV is one way that you can use to visualize your alignment and I believe, has anyone ever used IGV before? Okay, that's great. So you can simply just load the ban file in IGV and the way it works is that you get this window where you have the ideogram, so that's like the chromosome, the P-arm, the Q-arm and the chromosome along with the bands, you have the centromere and in the bottom you can load a gene track so that for a specific gene, it will tell you the exonic and entronic regions, you can change the gene track, you can add whatever indication that you have or you choose to use. And then you get the reads. So this is a pile up of the reads that you have sequenced and this plot right here shows you the distribution of the coverage for the exonic islands that you have. The lines that connect these red fragments, they represent the entronic regions that the aligner or top hat predicted based on the reads that you have. So what you try to do is you try to look at the predicted splice junction which are these blue lines, light blue lines and then you compare them to the known annotation to see if the islands of exons and introns match. And they should most of the time match unless you are dealing with novel genes where you have novel splice junctions that might not be annotated in the database. But most of the time the splice junctions that you detect are probably known and they should match the transcripts at the bottom of the screen. Now you can color code the reads as well. You can color code them to tell you whether they're coming from the first read or the second read. If you're doing strand specific libraries then the color coding is very important because it tells you whether it's coming from the sense or anti-sense. So it adds another layer of information that can be useful. And you can look at the color of the reads and you can look at the direction of the transcription. You look at the arrow and that will tell you whether it's coming from the sense or anti-sense and if that direction actually matches the color of your reads if they're coming from the same strand. So here are some other alternatives to IGV. I personally haven't used any other visualization other than IGV. And I just use it, I don't know, like I don't, it's really hard to pick a region because the transcriptome is so huge unless you're interested in a specific gene if you're doing targeted panel or after you actually do differential expression and then you find a list of genes they're interested in, then you'd go back and just make sure that the coverage is okay and these two. Otherwise it's really hard to assess the whole transcriptome using IGV. It will just take forever and it's just not efficient, not an efficient way of assessing QC. And that's why you should use tools to assess the QC for you. And here's just a list of metrics that you can look at to assess the QC of your alignments and I'll go through each one of these metrics and I'll show you a plot that you can generate to help you understand each one of these metrics. So we're gonna be looking at the coverage bias, we're looking at the nucleotide content distribution, the base quality, the read quality distribution, the PCR artifact, the depth, the sequencing depth and how determined, how deep should you go when it comes to sequencing. You can look at the base distribution and finally the insert size distribution. Now all of these plots that I'm going to show you today they are coming from one tool called R6C. You are not obligated to use that tool. You can go ahead and use whatever tool you want. You should adjust some guidelines of things or metrics that you can look at. You can establish, you can write your own script to pull these metrics or you can use a combination of other tools. I believe Picard has a very nice tool for RNAC, collect RNAC metrics, I think that's what it's called. So you can ideally you would like to combine multiple tools when you're developing a pipeline for your QC, but this is just a good starting point for QC. So the first metric that we'll look at is three prime and five prime bias or you can think of it as coverage uniformity. So you want to look at the transcripts and you want to make sure that the coverage across the transcript is uniform, that both ends of the transcript are being covered the same way. Now, in order to calculate that what R6C does because the transcripts they have different lengths. So it takes all the transcripts and then it splits them into quantiles or bins from one to 100 and it looks at the coverage for each one of these bins and that will help you visualize. So if you plot on the right, we're looking at a plot where the X axis represents the quantiles from zero to 100 or you can think of them as bins from zero to 100 and then the Y axis is the coverage. And once you do the plot, you realize that there are two groups of samples. So there is a group of samples that seem to have consistent coverage across all the bins from the five end to the three end of the transcript. But you see a group of samples that seem to have a lot more coverage on the three end compared to the five end. Now, this is alarming because it could mean multiple things. It could mean that the transcript is actually, or your RNA is degraded. The five end has very low coverage compared to the three end. So if that's the case, you wanna go back and check your RIN number and see if it's low or if it's high. It could also mean that there is a three prime bias, maybe the kit that you use, maybe it's poly A selection. If you're doing poly A, then you're only pulling things that have from the three prime end. So if that's the case, you wanna go back and then maybe look at assessing the library selection that you've done, choose another one or so on. If you don't adjust for these differences, they will actually have some bad consequences in when it comes to expression estimation. Because if you have bias in your coverage, you will end up underestimating or overestimating your expression, depending on how severe the bias is. Now there are methods, computational methods, where you can correct for these. If you absolutely cannot obtain new samples or you can't rerun those lanes, then you can correct for them, but you have to be aware of them and figure out why and then correct for it before you perform expression estimation. Any question? Yeah, I just wanted to ask. How you can correct for them? Yeah, occasionally, unless you- Yeah, I don't have a specific tool on the top of my head that I'm aware of, but I guess it really depends on the severity. Like if all your samples have that or if you've done two groups and one group does, maybe you can use that as a benchmark to normalize and try to correct the distribution based on the other group that doesn't have that artifact. But at the end of the day, if you're using a downstream, you will see the byproduct of this bias somewhere unless you adjust for it in your downstream analysis. So I'm not sure how good the tools are at correcting those. Ideally, you would like to rerun the sample because it could be that you're missing out on other. If it's a polyase selection problem, then maybe you're missing out on reads that are not here. So even if you correct for them, the reads that are not there or transcripts that are not there, false negatives. Yes. In which group do you have the reward? Yeah. Is that a read one or five prime? That's a three prime.  It's five to three, I believe, five to three. Yes. For the genes? Yeah. So if the read aligns something, you have the coverage over the entire RNA or you have a coverage over at the end of the RNA. Yeah. The counting should not change, right? The counting should not change. What do you mean? I mean, the counting should be distributed across all the exons within. So there shouldn't be a bias towards one end or the other. The gene expression. Yeah. So you are counting the reads which are aligned into one set of genes. Yes. To the gene. Yes. So if it aligns at the five prime end or it aligns to the three prime end, the count should be the same, right? So it should not affect the process. If you are doing gene expression analysis, if you have this sort of sample distribution. Well, why are they not aligning to the three prime? Like you're saying if it's biasing towards one end, that's okay because the count is still there. Yeah. But the count should be distributed evenly because what if you are, if there is a degradation in your RNA, then you're biasing towards the short reads or the short transcripts, their expression will be higher than long transcripts depending on how studio the bias is. So for short transcripts, if there's more accounts on the five prime end or sorry, the three prime end, that's overestimating the expression versus very long transcripts where they'll have a lot of reads on the three prime but there's nothing on the five prime end. It can't be okay for just you to bias this consistency across all of your samples. Yeah. And sometimes it is. Yeah, and you can't guarantee at the selection process if you've missed any. It's a sign that you might have missed and that you can't really correct for because you didn't really pick it up if there was degraded. If it's yeah. Well, as I say, you need to figure out where it's actually happening. Like if you can go back and check your RIN or like the library quality to see if it was good or bad, if it was really good, then it's a sign that it wasn't maybe the library. It was something that happened after the library construction. So try to trace back the error and see where. You can produce a plot like this and if you set it. Yeah. If they're all the same and it doesn't look good. Most of the time when I've seen this, it was an indication of a kit. It was just the kit that was used. It was a reproducible error because of the kit that was used, so kit differences. We're pulsing it in something that was called an RAC client. Do you observe the similar pattern and write what we don't want to do, sir? Write what? If she is good, she should get a pretty even. She shouldn't bias you towards one end of the transcription. If it was zero, you would check the nature of the results. Yeah, a song, but I don't know that you would see that. Unless it's degraded. On this, unless it's. Another metric that you can look at is the nucleotide content. So here I'm looking at the position within a read. So assume that our read is 35 base long, very short read. And then we're looking at the nucleotide frequency at each position across all the reads in your library. And what you expect is that you expect the same distribution. So AC, GT to be about 25% presentation in your pool. However, when we do this for some of the alumina samples, you notice that there is a deviation from the expected 25% at the beginning of the read. And it seems that it's due to random primers that do the reverse transcribe RNA fragments to CDNA or double stranded CDNA. They seem to cause, they seem to be not very random and they cause certain patterns over the first few bases of the read. Now, this does slightly affect the alignment. So when way you can improve the alignment is by trimming or trying to adjust for those changes. I usually just trim the first bases of the read and then use the rest of the read that has AC, GT about 25%, 25% yes, it's always the beginning of the read. Beginning of the read, yeah. How many bases? Sorry, how many bases? Between zero and 10. I don't see much variation because of that in this sort of. But before you trim, do the plot, see if you have such distribution and if you do then I'll try to fix it. R6C, yes. R6C is the same? No, this is different, yeah, yeah. So there are multiple tools. There's that, there's also the card, collect RNA-seq metrics. And they don't all overlap in terms of the QC, so each tool would have some unique metrics and plus and that's why I said the union of all of these tools would be great if you can run all of them. Another metric you can look at is you can look at the distribution of the base distribution across the read. So here we're looking at the same read 35 bases long and we're looking at each base and we're looking at distribution of the quality. The quality is usually reported as a fret score and fret score is just the probability that the base calling is wrong. So a fret score, if you see a fret score of 30 or Q30 means there's one in a thousand chance that this base calling is wrong. So the higher the Q score, the better the quality of the base because it's less likely that it's an error. You notice that, so here most of the reads are, most of the bases are above Q30, which is great. You do realize that there, notice there is a decline in the quality. That's okay because that's just an expected for all the sequences by synthesis technique that the error profile increases as you go towards the end of the read. Just make sure that the overall distribution of your reads are above Q30. The, another metric that we look at is PCR duplication. So in DNA, I don't know, you guys didn't touch upon PCR. Did you talk about the collapsing, collapsing your data? So in DNA, you collapse the data because some of the reads can be PCR artifacts. So you expect the reads in your genome to overlap. You don't expect them to pile up in a one region. That's when we talk about DNA when we talk about RNA. Things are a bit different because the start positions in the RNA, they're not random. They're the genes they have start a transcription site where they start. So there is a likelihood that you will see reads that pile up at a certain position. So you can't really differentiate between a PCR artifact and a true biological transcription signal. So I usually don't do collapsing when it comes to RNA. However, if you want to assess the duplication rate, there are various ways you can do that. So one way that RCC does it, it looks at the sequences and it looks at the sequences for both reads, read one and read two. And it looks at the position. So they must have the first start point for read one and end for read two. It also looks at the sequence context. So the sequence content is exactly the same. Then it calculates the duplication rate based on the sequence content and based on the start and end and it will give you an estimation of that. And based on that, it's up to you if you see a very high level of duplication, then you can go back and check why this might be happening. Is it the library or prep? And then you get to choose whether or not you want to collapse and check the dynamic range of your expression after and before collapsing to make sure that it doesn't change massively. Sequencing depth. So if you're trying to figure out how many lanes of sequencing you want to do for your study, how many samples you want to multiplex in one lane, again, that question is dependent on so many things. It depends on what kind of sequencing you're doing.  What kind of analysis do you want to do downstream? Are you just doing variant calling from RNA? Are you doing expression estimation? Are you doing fusion? So there are a lot of variables that you have to consider when you're trying to decide how many reads per sample, but one simple way can be by doing a pilot experiment where we take one of your samples and then run a full lane on it and then do a saturation experiment or a saturation test. So what the tool does, it takes the aligned band file and it samples at different levels. So it samples 10% of the reads randomly, 20%, 30%, 40%, up to 100%. And at each sampling level, it calculates the number of novel junctions and known junctions based on a database that you provide or like a file that you provide. So it goes through them and then calculates the number of known and novel junctions and then it comes up with this plot. So the point where you want to stop sequencing is the point where your total junctions have saturated. So no matter how many reads you're adding, you're not actually getting any more information. You're not getting any more junctions. Now you notice here there are three curves. So the blue one represents all junctions. The red one represents the known junctions and the green one represents the novel junctions. And you will notice that the first one that saturates is the known junctions. And that's because there are a lot of false positive junctions that you'll get with whatever tool you're trying to do. You will get false positive junctions, they're novel junctions. So you can consider both a novel and known and try to come up with a threshold at which you're not getting any more junctions. Now you can do the same thing that's not an R6C but you can also look at maybe the expression of gene families. So you do the same sampling experiment and see how many more genes are you picking up? How many genes are you picking up by adding more reads? And at a point at which you saturate where you're not introducing any new genes or that the expression doesn't change, then maybe that's the point where you can stop.  Base distribution, so what this is doing is just taking the bases that have been aligned and it's trying to figure out where in the transcriptome those bases are aligning, are they in the intronic region, exonic region, intergenic region, and so on. And that will confirm the type of library that you've used. If you use Total RNA, you'll obviously see more intergenic regions. If you use PolyA selection, then you should see a lot more coding or exonic bases. And if there's any discrepancy, then you wanna go back and check why. You can also look at the inter-size distribution. So you have your fragment is what you're trying to sequence. This is the fragment of CDNA. You attach the adapters to it before you sequence it. So when we talk about inter-size, we're talking about the fragment without the adapter sequences. When you're talking about the inner distance, that's the distance between the two reads. So if this is your CDNA fragment, you're trying to sequence read one. So that will be like 100 bases this way, 100 bases this way. Whatever is left that hasn't been sequenced from that fragment is called the inner mate or inner distance. And the reason why I'm mentioning this because we'll use that metric in the integrated assignment in a bit. So keep that in mind when we're doing that. But you can use that information, as I've mentioned, you can pull it from the cigar string. Or sorry, you can pull it from the, there's a column that has the distance from read one to read two. And that information can help you come up with an inter-size distribution. So that will approximate the fragment size of the library that you started with. And then you can check it against what was reported or what you were told and see if the library size matches or not. And I believe also, you can also use IGV. If you have a specific gene in mind, a specific site in mind, you can load your data in IGV. And if you're looking for specific variants, you can, the variants or the SNPs, they will be colored. If any base that does not match the reference will be colored. So you can look at that. And if you want to calculate the allele frequency, you simply just count the number of reads that have a different base over the total number of reads that cover that site. And that will give you the allele frequency for that variant that you're interested in. However, this is just if you know what gene you're interested in with Exxon. Ideally what you want to do is some sort of variant calling tool or you can do SAM tools pile up that goes through every single base in your genome. And it does that for you. It calculates the number of bases that match the reference, number of bases that are alternate, and it will calculate that frequency for you at each site. Okay, so that concludes the second module.


and a good evening. You can go to love in France, but you cannot become a Frenchman. You can go to love in Germany or Turkey or Japan, but you cannot become a German or Turkish or Japanese. But anyone from any corner of the earth can come to love in America and become an American. We'll come back to a nation of immigrants, a bi-weekly interview program featuring the lives of the immigrants, knowledge, diversity, and inclusion. Created by Sing Tank Hawaii, Kingsfield Law Office, and U.S. China Cultural Media Group. Our guests share their life stories, journey to the United States, and their contributions to cultural diversity. Today's guest is Vivian Wu, founder and the CEO of Mighty Voice, Dasheng Media. Welcome, Vivian. Hi. Hi. Nice to see you on the screen. Thank you for having me here. Our pleasure and honor. Thank you, Vivian. You are my favorite investigative journalist, but now you are a founder and the CEO of a company in New York. You are a seasoned media industry professional with 20 years of experience in journalism, international reporting, and media innovation in both English and Chinese markets. Currently, you are the founder and the CEO of Mighty Voice, AKA Dasheng Media, a New York-based cultural and creative content production corporation. At Mighty Voice, you lead a global team of media professionals and tech experts spearheading the development of groundbreaking model of community journalism. It's a very impressive resume, Vivian. Even I know you're very well, but you have constantly surprised me with your innovation and your adventure. And first, could you share with our audience that the pivotal moments that led you to relocate to the United States and establish a company in New York City? Okay. Thank you for the nice work, Chang. You know me well. So put my background very briefly. I really spent like 20 years in the media field, but I spent years in newspaper. I started my career from 2003 when China was actually seeing a very prosperous, very inspirational period of journalism production, market change, and, you know, progressive era. So I was lucky that I did a lot of story. I accumulate a lot of skills, but I changed my job several times. I moved from Beijing to Hong Kong, from Hong Kong to New York, and New York to London. So after years of working in the field, I realized, okay, I'm lucky that I accumulate extensive experience. But I also see the change of media landscape, change of technology, change of people's habits. Actually, there are many pivotal moments. I see how people's habits of absorbing information was innovated by the application of social media. Also, it was changed by the TikTok, a short video on YouTube. And at the same time, you see the diminishing of high quality content, especially if you, like me, I work as a bridge. It's like I try to tell China's story to the world, but also I try to bring the global perspective to the Chinese audience by switching the languages. So I feel like, okay, there are several moments. I feel, okay, first I really need to do something to change the vision, to change the vision about media shared quite about public. Also, I need to, I realize too long in the institutional media, you'll find on one side there are a lot of online celebrities, a lot of individual voices. They might not be very professionally trained, but they have a chance to voice out. But in the meanwhile, the public suffered the deteriorated quality of content and there was misinformation, disinformation, polarization of public opinions. People refused to talk to each other. On the other side, you'll find, okay, there are professionals, but they don't have a chance or platform to use that to better help the public to make better decisions. So this is a professional thinking pattern. And of course, in my personal period, in my personal dimension, the pivotal moment once was, I see China Xi Jinping changing his constitution and China basically publicly announced that they don't need to learn from the outside world anymore. They have their own system of value system, professional level system, even definition of democracy and freedom, etc. So on the other side, you'll find, okay, Trump became a president and then there are a lot of drama, a lot of protests in Hong Kong. The world needs more rational voices, but also they need a more individual platform that can address individualized needs. So all this, all these facts, elements, historical changes forced me to think, okay, I'm at a pivotal juncture. I need to change that to harness my accumulated skills while embracing the freedom that can empower me to innovate and address more specific needs, both from the public, but also by myself. So I don't know if I answer a question, but it's like a long journey in the media field, how technology changed people's lives. You see how the public suffered, you know, Western quality of journalism, the public needs a better way of doing journalism publication, etc. And you also see the social political changes, both on the two sides of the Pacific Ocean. You see, okay, it's time for change, but where to start? Let's start by relocation, start by doing a new platform that I can somehow design, control, also fast to develop without any baggage I see in the institutional media. You answer the question very well, both professionally and personally. I'm particularly intrigued by your mention of the quality content. Now we are living in this very saturated information time and bombarded by TikTok, Instagram, YouTube, Twitter, you name it. But the need for quality content couldn't be more urgent. And so you primarily gather your insights and updates, where do you gather this information? Because when I work with my students, I always ask them that, where do you get your information? And tell me the primary sources of your information, is traditional media, print, digital, or social media. And so my question to you as a professional, obviously, is this question more relevant to your work? So where do you primarily gather your insights and updates about ongoing developments, both in the United States and in China? And the second question is, how do you ensure the reliability and the integrity of those resources? Obviously, you have your judgment about what considered reliable sources, but sometimes even mainstream media, they've been confused by misinformation and disinformation. A very relevant and important question. Actually, when I was working as a manager at BBC, if I was doing recruitment, we must make a question, what do you read, and what's your source? So let me share how I start my day and how I form my reading habit. It's like, it's very, very important first to sit in the middle, and you need to make sure that you have diverse sources of information. That being said, it's actually hard, because as you said, people confuse. And usually, if you are not professional, and you don't have a checked list of verified information, reliable source, it's very, very easy for you to trust, mistrust something, and it's just retweeted or repost it either on WeChat or Twitter. And actually, you are part of this relay of misinformation. And then we see actually wrong information go viral. So how to make sure that I read good information? I usually, I spend, I start my day, first I have many, many apps installed on my phone, but I have two phones. I have one phone following the China's apps and and your related Chinese or traditional Chinese, because I also monitor Hong Kong, Taiwan, and domestic Chinese media outlets. So this very diverse source of information can help me to verify any information if it's to do with China policy, for example. But this is just a one layer of Chinese reading. And of course, I have, I followed many good quality, even though there's still a censorship issue, but there's still somehow verified source of big news or local news. For example, Cai Xin, Cai Jing, there are some, or Xinhua, actually, when I mentioned Xinhua agency, because there's a monopoly of official information. Of course, that's a fact that's suffered by the journalism industry. That means, all the authentic information to do with top authority or governmental behaviors were not monopolized by the state's media. So even though you don't like the narrative, you still need to follow what Xinhua has said. Or a lot of information was released by CCTV or their state-owned outlets. And you also have a layer of local news. And they have their WeChat accounts. They have, but don't forget, there are actually a whole range of public intellectuals or journalists or retired journalists that they have this effect checking habits. So you always need to cross-exam one single information. And if I see a piece of story on any platform, I try to find three more sources. And I always try to find the original source if that story was quoting somebody's facts from that original source. English side. And then this is the first one or two hours of my day. I just have a navigation what's really going on. And then I need to quickly decide, oh, if this is Chinese news, how the world will see this. And of course, in the meanwhile, in the parallel, I actually have apps installed on my another phone, American phone. We have CNBBC, New York Times, New Yorker, Economist, FT, you name it. And also, you need to follow verified accounts of journalists, the same tank, all the government's official accounts on Twitter if they have. And of course, always, always try to find the original source of that fact. So what I'm saying here is people need to, as individuals, even though you're not professional, you need to have this habit of fact checking. We are suffering from this echo chamber problem. Especially this echo chamber was enhanced and was actually repeated by the social media. That means if you just follow 50 people in your circle, then you are always taken for granted that, okay, if this story is shared by my friend, without any hesitation, I'll be thinking, I will repost it. But as I said in the beginning, you are then probably part of this disinformation redistribution game. So always, always double check, even though this information is shared by your neighbor, only if that neighbor is your, is an investigative reporter. But even though you still cannot take for granted, that's true, you still need to double check. So what I'm saying is like, if you have this habit of cross-examining, always, if you read several languages, it's always important to check the source from the original country's local verified media outlets. And then if you could, check with your local friends. And then I think then you will have a very clear navigation of what's really going on. And then of course, we talk about the topics or other stories that as a media professional needs to address. Yeah. So this is just a way of thinking and having. Yeah, I see. I found a lot of, thank you for sharing the very insightful observation and some of the professional workflow of gathering information. I found some similarities between our works that when you always go back to the original source and for the legal practice, and we always start, we can start with the secondary sources, but at the end of the day, we must go back to the primary source. And always any argument must be substantiated and any evidence must be corroborated. And I also intrigued by you mentioning of the parallel universe and echo chamber, also known as echo chamber, but it feels like inevitable and we are living in parallel universes. I'm not only talking about people in different countries, geography, living in different parallel universes, but also even you and my neighbor and I can live in parallel universe. And it's the same family member in the same household. If one is follower of Fox News, the other is MSNBC. Obviously, two of them live in completely different universe. And so my question, you are leading Russian media and the mighty voice, and it just stands out pretty quickly. I congratulate you for marvelous, your remarkable achievement in a short period of time.  So there must be challenges to you. There must be obstacles and how to stand out, how to, you know, the subtitle of our show today is Mighty Voice Be Heard. And you want to be heard and we hear you, but how can you ensure that your company can distinguish from other media outlets and continue to engage and the audience. And basically the question at the end of the day is how can you make your voice well heard and distinguished from other voices? Yeah, you're absolutely right. I think our biggest challenge is actually people's attention. It's a computation for attention. People are busy with their social media, chatting, messaging, applications. It's very hard to drag people out of their comfort zone and grab their attention. So how to do this? I think first, even though the market is busy, but you see a lot of professionals giving up their jobs. I mean, I'm saying this, we see in America or a free society, there's a continuous layer of professionals. Media industry is fading off because of the high tech, because people's change of, you know, change of people's habits of grabbing information, people would rather use social media than reading the high quality professionals journalism. So what I'm also, this is even more important and more prominent in Chinese sphere. So I've done both in English-Chinese. I decided we have three strategies. First is we give a very refreshing, very strong, prominent branding of our values. So our mission is we create a space for freedom, decency, and the beauty. It sounds interesting, but also different because we don't talk about freedom speech, we don't talk about democracy. It's not the cliché you will find from the international media or no local Chinese media will talk about the freedom openly like us. So we're afraid that means we don't have self-censorship, we don't have censorship, we uphold the freedom that we really take it seriously. It means nobody can, you know, nobody can shake up our independence. And we highlight, we are very, very very strict with our quality. That means we are very carefully, we're very carefully choosing the topic of our stories. We are very strict with quality. You see the professional image, design, narrative, fact-checking. We also have very comprehensive and aggressive social media strategy, even though we have a very, very small team, but we have this young kids, young people who are very dry, who are very driven to do something different. They are bored by the status quo. So we are very aggressive, also very diligent on social media. And then people immediately get, oh, this is a very refreshing image. And look at their stories. Wow, the stories are very relevant. We address most important issues not only in the free world, but also in China. And we also highlight our global vision. This is so important because imagine China is now getting more and more close that they are more isolated from the outside world, but actually demand for, you know, global vision of narrative, a wider range of topic selection. And also like Qing Liu is like selected, sophisticated, intellectual. But we don't sound arrogant. We are very relatable. We are very approachable. Even our design, our branding is designed by young girls. She's universally beautiful. She has a very good artistic sense. So we try our best to stand up to give you a sense of this is a decent, free, high quality professional platform that you can be yourself. Because we are not arrogant. We make sure that all the topics we choose are relatable. We try to echo people's, you know, the soft points, your pain points. Then people need such a new platform. People need such new voice. People are very bored by unrelatable, somehow arrogant, too remote address of China issues if you read international headlines, even though they're good stories, but it's just not that approachable. Or people are bored by the trash on social media. We have to say this. It's like a talk change people's habits. People are very impatient, but high and good quality content actually is proven to be needed. Then I'm trying to address that need. So now I can be very proud to say Dasheng really has very good reputation among our professional peers. And I also want to say it's like, I am pretty sure every tweet, every story, every video we produce, you won't, as a reader, as an audience, you won't be regretful if you share that. You will be proud to say, hey, let me recommend this story. Read it.  So if you feel proud of something, of course, you will be very enthusiastic sharing it. It's what I see every day. It's like I'm approached by my friends, my audience, my readers, they say, great, Dasheng,加油, add oil, do it well. So I don't know if I answered your question. You did. Splendid, splendid. It's your voice, Dasheng's voice is very refreshing. And in the current media landscape is such a pleasant surprise. And it's so reassuring to see that you are bringing fresh air to the media landscape. I really appreciate that. And we are almost running out of time. But I do have so many questions I want to ask you. We have to invite you back to the show. But let's switch to a lighter side. And I have three quick questions for you. The first one is, who is your favorite journalist? And worldwide, do down the limit to the America or in China? Second question is about time travel. If you're a young lady, but if time travel permitted, you could travel back to your early 20th fresh out of college. What wisdom you could share with a younger self? And a lot about the not list. Is there a particular book or a TV program or movie you recommended to our audience? Three quick questions, please. Okay, all great questions. Let me try to answer. Actually, for the first question, who are my favorite journalists? There are too many. Because remember, I spent 20 years. So basically, I need like a constant reboost. I need a constant push in my back. And I feel motivated. But I can name several like of course, you know, Ariana Farachi. Farachi, you know, Ariana Farachi, she's a role model. But the more the older guys, especially the more I am driven to be a female leader, I realize she's a very, she's a badass. She's not afraid of being tough. But also her pieces, she's never afraid of challenging the strongest and the toughest, but she always tried to get the questions out. So I'm always inspired by her, both for the technique and the, you know, personal style of working. But I'm not that tough as she was. I'm not that tough. I'm trying to. So also like a journalist from New York Times, such as Chris Buckley. Oh, yeah. He's so good because he's so diligent. He's studying Chinese. He always sends some sentences out so well. In English, you'll see Bingo, this guy knows China better than me. So you then, you know, also she he, if he was writing for some obituaries, for example, he spent the years studying one persona. This is very special, very original, inspirational. And for the book, of course, the first book came into my mind, George Orville's 1984. Yes. Never gets outdated. Never gets old. And I somehow realize, is this a function, is this a fiction or nonfiction? It's nonfiction. It's nonfiction. It's nonfiction. And for the movie. Time travel. Time travel. If I was talking to me back to 20 years younger, I would say stick to your stick to the principles, stick to the values. You're, you're right. You're not wrong. It's hard to be different. But if you carry on your C by the other side of your journey was C, you're probably one of the very few, but you're right.  Yeah. Yeah. What wonderful answers to, to the short questions. And thank you so much for the time of the end. Again, as I said, I've been constantly amazed by your achievement and by the fresh air you bring to the media landscape. And I will continue to, to watch your accomplishment and congratulate you for the new website and new media outlets you have achieved so much. As we said in your New Yorker, I'm still a Minnesota, but as we said in Minnesota, be well, do good work and keep in touch. Thank you, Vivian. Thank you. My pleasure. Thank you for the time. Thank you for the nice work. Thank you. Aloha.


This study investigated the effects of whole body vibration, WBV, therapy on gait dynamics and chronic stroke patients. A total of 64 participants were recruited and randomly assigned into two groups, one receiving routine physiotherapy, RP, and the other receiving WBV therapy. Both groups received 12 sessions of their respective treatments over a period of two weeks. The outcomes measured included the timed up and go, TUG, test and 10, meter walk test, 10MWT. The results showed that WBV therapy significantly improved the scores of TUG and 10MWTSS while there was no significant change in 10MWTFS. Additionally, the WBV group had better improvements than the RP group. This suggests that WBV therapy may be beneficial in improving gait dynamics and chronic stroke patients. This article was authored by Hafiz Sayed Ijiz Ahmed Birk, Hossein Karimi, Ishfa Kamad, and others. We are article.tv, links in the description below.


We looked it up to see what he looked like. In the pictures, everything that we looked at, he did not have long hair. He was basically shaved bald and had hair back here and feathers. He was a whole lot more intimidating looking, but from what we were told from the history of this statue right here, the whole purpose of them creating this way is they didn't want people to be scared of what he looked like or be intimidated by him so that's why they designed him what they did.