Welcome to MOOC course on Introduction to Proteogenomics. In the last few lectures, the emeritus speakers Dr. Kelly Reggels and Bing Zhang talked about genomics, gene mutations and its effects on phenotype. So, now we have better understanding of the importance of mutations in a clinical study and level of impact it can make in the efficient treatment therefore, now you know the next logical way is how to do these experiments using the latest technologies available especially how to use various type of NGS instruments to do these kind of experiments yourself. In this slide today we have invited one of the industry leader Dr. Atim Agrawal from Thermo Fisher Scientific and she is going to talk about how one could use the index generation sequencing technology and sequence targeted genes in the genome. In today's lecture she will talk about NGS technology evolution and development over the time and then there will be hands on session which are also going to show how to use these instruments for generating data. So, let us welcome Dr. Agrawal to talk to us about recent advancements in the field of NGS and data analysis with a short demonstration. Basically we are going to talk about the recent advancements which are happening in sequencing field like pre 1990 it was a very steady field wherein people were using radioactive labelled DDNTPs and that is how things were getting sequenced and then it was quite some time then like more than a decade wherein Sanger sequencing was the only sole sequencing technology wherein people were using fluorescent DDNTPs and they were sequencing various fragments. So, past decade there had been many advancements in terms of sequencing it is not only Sanger now there are many technologies which have come up in terms of next generation sequencing and how they primarily differ from a Sanger sequencing or capillary sequencing which all of us were used to for quite some time was that earlier it was more or less it was that one well was sequencing one fragment yeah. So, you could have loaded a 96 well plate or a 384 well plate and you could have got 96 sequences or 384 sequences. Now, within this past decade what has happened is there is a term called massively parallel sequencing which has come up. So, massively parallel sequencing is something wherein you are sequencing millions of fragments at one go. So, that is what you are trying to do and because you have been able to do that those millions of sequencing together now you are trying to solve a lot of bigger problems faster. So, that is how like you would know that a human genome for the first time when it was being done on Sanger it took around 10 to 11 years now things are being done in 3 days. So, that is how the technology has advanced and likewise even the cost at which you were sequencing the genome earlier and the cost at which you are doing now that is also tremendously decreased. I coming from Thermo Fisher I am very I would say I am very proud that I was all these decades my company was associated with these sequence sequencing technologies and we had been pioneer in this arena. So, I would just like to show you very. So, this is this is how our Sanger sequences look and where you are basically plating either a 96 well plate or a 384 well plate and you generate sequences. Here also we have come up with various advancements wherein sequences are now cartridge based. So, the user only has to put in a cartridge and he is good to go and he is just has his prepared samples. This is what is we are here to talk about today next generation sequencing wherein you have thousands to billions of reads being produced per run and the data which you are producing is in terms of MBs and GBs. These sequences can be like applied to various many different fields like metagenomics, microbial sequencing, RNA-seq, exome sequencing, transcriptome sequencing, targeted sequencing. So, all of that a basic workflow what is involved is I will just briefly explain this slide. So, this is the sequencer which we are here to which we are here displaying and this is ING studio S5 which has five different chips which gives you a versatility from producing anywhere from 2 million reads to 130 million reads that is a very big like very important feature. So, that like if you have this instrument in your institute a person who wants to do microbial genome sequencing or who wants to just sequence a metagenome he can also use the same system and a person who wants to do a full exome or a transcriptome he can also use the same system. The other big highlight of our system is that one is it gives versatility that it gives you a various like it gives you a huge span in terms of the number of million reads. Another big advantage specifically for oncology is that with oncology a very big problem is in terms of the amount of sample you are dealing with. You are generally dealing with tissue blocks which yield very very less amount of DNA or RNA as a starting material. So, that ways we are we have a very robust technology called amphi-seq technology and this technology has the capacity to multiplex many hundreds and hundreds of targets at one go and amplify all of them at similar efficiency and then sequence them all of them at similar efficiency. So, then you are not losing on. So, supposedly you want to study 100 targets out of a tumor which is some very few mm and you get hardly 10 nanograms of DNA or nucleic acid material. Now, you want to study 100 targets. So, this amphi-seq technology is basically making it is such a robust technology that all these primer pairs which are working in one tube they are so efficiently being mixed that all these targets are being amplified at this similar efficiency and that is how you are sequencing them at a similar efficiency. So, that is what we are we have pointed it out here that this sequencer along with its technology has the lowest DNA or RNA input requirements. Then these are the three systems which Thermo Fisher provides Gene Studio S5, S5 plus, S5 prime and basically they differ in their compute capabilities in terms of their throughputs and in terms of the compatibility of chips they differ very in a very minor way. But other than that you have this 510 to 550 chip which can be used with these systems and the difference in compute power as a user what it is giving you is faster run times. So, a user a very new user is good probably with S5 wherein run times really do not matter to him much. An established cruiser might would like to go for an S5 plus who has some planned experiments one by one and then he just wants he does not want to get held with the time of run and then this is S5 prime wherein you have back to back runs and this is the technology which has the capacity to finish a run within as little as even 2.5 hours. So, this has been on the in this research arena or clinical diagnostic arena for since around 2010 this technology and it has around 4500 publications all over and since we have been focusing a lot into clinical oncology and all. So, maybe for us it is more of application notes and all lately and certain studies which have been done and published in different formats other than scientific publications. So, the workflow is what we want to show here is so, a typical NGS workflow would start from nucleic acid extraction. So, this nucleic acid can be a DNA can be an RNA. So, once you have extracted this nucleic acid then you prepare library. Now, library preparation is nothing whatever DNA or RNA you have extracted basically it depends on what kind of application you are working on. If you are working on microbial sequencing then probably you are starting sample is a viral DNA or RNA or a bacterial DNA and RNA and you want to sequence that order at one go. So, what you are going to do in that library preparation is that you are going to share this genome either by enzymatic sharing or by mechanical sharing you break down this genome into small pieces and then you like it adapters these are double-stranded fragments which are ligated on both the ends and that is what you do like basically what you are trying to do is you are sharing down the interested genome into smaller parts and ligating it with adapters. The sequence of these adapters is known. So, that is how the primer can sit and it can sequence the region in between. So, that is what you do in a library preparation. Now, for an oncology sample wherein you are trying to look for different markers specific for a particular cancer or a particular type of cancer what you are going to do is you isolate DNA and RNA and we have wonderful panels which are like spanning from we have panels for solid tumor, we have targeted panels for human ecology, we have panels for cell-free DNA or RNA that is liquid biopsy which is gaining a lot of attention these days then we have panels for addressing the immune oncology also. So, you use any of these panels you amplify the targeted portions and then once you have prepared this library what you do is this is your template preparation during this part what you are doing you are amplifying these library molecules onto the ion spheres. This is being done onto this system wherein what you are doing is this these are two tubes wherein in one tube there is oil and in one tube there is a recovery solution or some kind of a detergent.  Then you have an enricher you have subsequent slides wherein I can tell you the biology of it you have an enricher wherein you have an 8 well strip kind of a thing placed and what you do is that your amplified ion spheres because not probably not all of the ion spheres have amplified so what you are doing is that you are placing that mixture here when you were amplifying you were using a biotinylated primer. So whatever has amplified whatever ion spheres have amplified they have now biotinyl label on them so what you do you place the streptavidin coated beads in one of the well and this is again a senai automated way this system this is called enrichment system this will go on its own for half an hour and then finally through this biotin streptavidin binding it will fish out all the amplified ion spheres and it will leave the non-amplified ion spheres and once that is done I will just cover that part in the slides first. So we have two solutions one is the semi-automated way which we have presented here we can show you the videos for the fully automated way of sequencing wherein you use a ion shift so wherein you do not have to deal with one touch two and enrichment what you do is you this ion shift this serves two purpose one is it can prepare libraries for you and second is it can do the all the amplification on these ion spheres and you get ready to load it ready chips which are already being loaded these chips these are basically silicon wafers on which there are millions of wells these millions of wells are basically enabling these millions of fragments to be sequenced all at once. So once so what happens is on the system so this is the once you have your sample prepared if you are using ion chef then your sample is automatically loaded onto these chips if you are using the semi-automated system then you have to load the sample with a pipette and once it is loaded this is the sequencer which takes not even 15 minutes to set it up so there is a there is a lot of apprehension that a next generation sequencer is very hard to kind of handle and it is it is difficult to manage that kind of a system that is what we want to show you that this is the chip clamp wherein the chip goes and sits these are already bottled reagents wherein if you see this is a sequencing cartridge wherein all the four DNTPs are being placed then behind this there is the waste cartridge this is the wash solution which goes onto the chip every time sequence polymerization has happened and this is the solution this is the wash solution after every sequencing run this wash solution goes on to the into the fluid lines and that is how it cleans the system. So now you can imagine how much time it would actually take you to pull these cartridges out of a aluminum foil and place it into the instrument the instrument on itself once you have placed all these reagents on the instrument so the instrument takes around 40 minutes to initialize itself and then you you already have your loaded chip here and you are good to go and this is the fastest system which can produce BAM5 which can produce data or which can finish a sequencing run within as little as 2.5 hours. So we have we I had shown you various models of the chip starting from 510 chip to 550 chip these chips have different capacities for an ion chemistry you can in terms of read lens you can go from a 200 base pair to 600 base pair yeah. So why these read lengths are important you consider different read lengths for different kinds of experiments. So supposedly your intention is to sequence certain markers from a tumor sample. So now a tumor biopsy as it is whatever DNA or RNA you if it is not a fresh frozen biopsy it is something like a FFP it is coming from an FFP block that DNA or RNA itself is highly degraded. So then you will use a smaller sequencing chemistry to sequence these small portions if it is from if your intention is to sequence a metagenome then you are trying to you are trying to deduce various kinds of bacteria or viruses present in that sample whether it is a sample which is coming from a cancer biopsy whether it is a sample coming from an environment whether it is a sample coming from a soil. So if you are studying metagenomics you would like to read as long as possible then probably you would go in for a 400 or a 600 base pair chemistry. So that is how things differ in terms of read length and what I want to point out is my colleague Harsh is going to talk about the data analysis part very fast. So we are not only leaving you at the sequencing part we have full-fledged solutions in terms of data analysis which takes you so in and further to for clinical people it also helps in generating reports. So reports which can be directly given out to patients and be told that okay this is the therapy which you could be using for treatment. So going to the next slide just these are just some figures of the workflow. So this is the library preparation what I was talking. So supposedly I am just taking an example of one of the assay this is on combined focus assay which has 52 genes these are all oncogenes and in the form of hotspot mutations CNV gains fusion drivers. So it is a very well known fact that if you are working with oncology and if you are working with cancer genomics you could not be only looking at single nucleotide variations or you could not be only looking at insertions in deletions you have to look if you are looking at genomic aberrations you have to look them in totality. So you have single nucleotide variations you have CNV gains or losses you have fusion drivers everything now if these are the kind of mutations you are looking at your starting sample has to be RNA as well as DNA because these two parts of the markers are going to come from the DNA and fusion drivers are going to be reported from the RNA. So what you do is you isolate DNA and RNA you reverse transcribe the RNA and then you have this pool of DNA or cDNA you take a primer pool now this is what is the primer pool coming from so on combined focus assay which is dealing with 52 genes. So this primer pool will be targeting all these 52 all the hotspots and CNVs and fusion drivers in these 52 genes and this is going to be one while and your sample which is anywhere from even as less as 1 nanogram goes and into this one while and all these targets all these start so these are basically 52 genes which are actually addressing as they are addressing around 1000 biomarkers. So all these 1000 biomarkers are getting amplified in a single tube and so this is that amplification happening once you have generated these amplicons because these primers are coming from conserved regions you partially digest these and then you ligate adapters. This is what is we discussed about library preparation that you want to ligate the sequence of interest to adapters wherein the sequence is known so that the primers can come and sit here and sequence the region in between. So and we can use barcoded adapters why we are using barcoded adapters so that we can use more than one sample on a chip. So you have seen the chips and so basically you would like to sequence more and more sample so it would depend we already tell you that ok on this particular chip for this particular panel you can how many samples you can multiplex that comes with our literature. So that is how you are using these barcoded libraries. So this was the second step which is template preparation which can be done on a semi-automated way you would use an OT2 and an enricher. When you want to opt for a fully automated way it is iron chef which we could not get here but we have a video we will show you that video. So what is primarily happening is these are your hundreds and thousands of library molecules which are coming from the amplified portion which you have amplified from the sample and then you like the idea here during template preparation is that this one library molecule gets clonally amplified onto the ion sphere. So that when this molecule is being read the signal is enough that it can differentiate noise and the signal. This is what is happening so this is an ion sphere which has an oligo which is complementary to one of the adapters of the which are linked to the library. So this is your library molecule which on both sides is linked with an adapter and this is the primer and the primer extends. So this reaction is on this plate and this is Peltier control. So all these small droplets which are being formed during the emulsion PCR they were they are working as individual PCRs. So these library molecules are then getting amplified and what is what finally you are getting is you are getting this particular library molecule all over amplified onto the ion sphere. Now you are using streptopidin coated beads on this system and that is how you are fishing out the amplified beads with non-amplified beads and this is the ion chef system which is a fully automated way into how you prepare your libraries. So when you are preparing libraries using ion chef system you prepare your DNA samples and you load them into these cartridges and there are various other cartridges which are put in different sections of the system and that is how the final you get a final library pool into one of these tubes.  So the chips are also loaded automatically and then these chips are ready to go directly onto the sequencer. So this is what we have seen so in the sequencer you have first the chip which goes into this clamp which is a temperature controlled clamp and you use polymerase and this polymerase because it works at a specific temperature that is why this clamp is temperature controlled. So you already have your sample loaded onto this chip then this is the sequencing cartridge which has all the four nucleotides the NTPs which are flown onto this chip in a sequential manner. This is the wash solution this is the discard which goes at the back of the wash solution. This is the wash solution which goes onto every time there is a nucleotide being sent to the on the chip then this wash solution goes and it washes any extra the NTPs left on the chip and this is that wash solution which is placed here which you can see which cleans the system automatically after every run. So it practically does not take a user to more than 15 minutes to set the sequencer up and get going with the run. So that's all so I've practically shown you you install the waste container wash solution cleaning solution and reagent cartridge you install the chip and you select the run plan and you are good to go. This system also does not need any standalone computer with it it can be connected with LAN and then you can operate on the system from anywhere. So what is happening in terms of sequencing? So as you had seen this chip has millions of wells and in each well what is happening is now this is supposedly an iron sphere which has amplified molecules onto that we've already added primer which is against the adapter then the sequencer what it is doing through this cartridge it is pumping various DnTPs in a very sequential manner which is already known to the algorithm of these software. So what happens is the moment there is a DnTP flown in onto the chip so supposedly we are flowing in an ATP as the first nucleotide. So now this particular action is taking place on millions of wells on the chip. So all those wells which have an iron sphere which has a T as the first base they there and this ATP will go and bind the moment there is a phosphodiester bond being formed is a hydrogen ion release this and because we did clonal amplification we amplified one library molecule all over the ion sphere so there is not one hydrogen ion release they are going to be thousands of hydrogen ion release which are good enough to bring a change in pH for this particular well. Each well has its own sensor so now the system now the software will calculate or while the run is going on the software algorithm is also going on and it is seeing that from the first phase from the first run of DnTP when that was flown onto the system how many wells showed a polymerization and in how many there was a change in pH which got translated into a change in voltage and that is how we get a signal. This signal is very well differentiated from noise and that is what gives you the accuracy in data. So what is happening is the nucleotides are flowing sequentially over a chip there is one sensor per well there is direct detection of natural DNA extension and then this phenomena is happening in millions of wells at one time. So just a cartoon to show this in a better way this is your ISP these are those two adapters which you have ligated during the library preparation process and this is the sequence in between which is which you want to sequence. So what is happening is that the primer which we have added which the system is already added before the sample is being loaded onto the chip the primer comes in anneals here there is a sequential addition of DnTPs and so a T will come in bind there would be a change in pH which will be converted into change in voltage and that would be read as a signal. Then again now what happened was now this system has flown in a DATP now the second base in question is G because they do not have complementarity this particular base will get washed off then this thing this sequential addition of DnTPs will go on and signals will be captured what do you think will happen at this stage there are two ways and we are flowing in a T signal will be double and that is how the system will know that there were two ways. So the signal will be exponential if there were had been three ways so that is how it will work and based on these signals you get finally a sequence which is then a sequence coming from that particular well out of those millions of wells. So after a two and a half hour run you have probably 80 or 100 million such sequences which now need to be analyzed. So this is how a typical run report looks that this is your chip this is the area wherein you have loaded the sample and this I will not go into these parameters and this read basically this is the gradation in terms of how good the chip was loaded this is 90% loading and finally it will give you some statistics now this is a 530 chip run and what we have done is we have run a liquid biopsy panel for lung which is targeting the variations which are there in DNA so that will include single nucleotide variations and insertions and deletions and this is the typical read length because so as you would all know that liquid biopsy is gaining a lot of attention because of its non-invasive nature because it gives the way it is being sampled it gives a better representation of the tumor because if you are taking a solid tumor it could we all know its tumor sample is heterogeneous. So what sample we are actually taking the DNA out whether that is representing the actual tumor position tumor condition we do not know so liquid biopsy that ways is a better way of handling and more so because it is a non-invasive way so it becomes a very good prognostic marker so especially when you are dealing with lung cancer cases you are not allowed to do biopsy many times so after the point of time or at times there would be patients wherein you are just not having that flexibility of taking any biopsy so there these liquid biopsy solutions are coming very handy this again is a panel which has some 22 genes or things like and these are all multiplexed in one queue and they are all getting amplified at one go so this is so you generated so many million reads and these are the read lengths because it is a selfie DNA as it is by nature it is a fragmented DNA you do not get DNA fragments more than 120 to 170 base space so you would like your primer pairs to be amplified very short regions so that you do not skip on that. So you have learned about how sequencing genome or a specific target gene sequencing has now become very easy accessible and affordable to many of the researchers which could facilitate them to dig deeper and solve many clinical challenges after understanding about genomics and looking at some of these hands-on sessions about you know various technology platforms used from the leading industry leaders from the thermo Fisher or Illumina scientific now we are going to move on to the next module which is on proteomics so the next module I will talk to you about fundamental concepts of proteomic technologies and then we will have other immunity speakers who will talk to you about current workflows using mass spectrometry based proteomics and how to do different type of tools and databases for proteomics analysis. So let us stop here for the genomics module and of course you know you got excited you have learned lot of new things here but you have also now got exposed to various type of publicly available databases and repositories which you can download yourself and now start implementing those using various tools available and showed in this workshop from which now you can address many specific questions and you can probably do many experiments even without having non-availability of even the technologies or the NGS system in your lab or in your own place you can still do many of these experiments and obtain some very meaningful information thank you.


Well, hello from the National Archives public programs and education team. My name is Missy McNat and I'm an education specialist in Washington, D.C. And welcome to the National Archives Comes Alive Young Learners Program. You can find the information about our future programs on the National Archives website, archives.gov under attendant event and on the National Archives Facebook page. This morning we meet General George Washington, portrayed by Doug Thomas, educator and actor with the American Historic Theater. Most people know that George Washington was the first president of the United States. But did you know that he was twice unanimously elected to that office by the Electoral College? And one of the reasons that he successfully was elected unanimously to the highest office in the land were his unparalleled leadership skills. So what makes a great leader? According to Forbes, a leader has the following qualities. Integrity, sincere enthusiasm, great communication skills, loyalty, decisiveness, and the ability to empower others. And I would add to that list determination, creativity, flexibility, and being a visionary. And General Washington as the commander-in-chief of the newly created American Continental Army had those skills and led the army through the, successfully through the Revolutionary War. And today we're going to hear specifically about the campaign to push the British out of New Jersey. So in the holdings of the National Archives are numerous records related to George Washington. A quick search in docsteach, docsteach.org brought up 213 records. So we're going to look at a couple of those today. And the first one is a portrait of George Washington when he was a colonel in the regiment in Virginia in 1772. So that was before the Revolutionary War and a support by Charles Wilson Peale. On the next slide, we see General Washington and his men crossing the ice choked Delaware River as part of the campaign to push the British out of New Jersey. And this is an engraving that comes from the painting by Immanuel Gottlieb Lloyds that was actually painted in the middle of the 19th century. Now we're in the next slide, we're going to skip to the end of the Revolutionary War. And we're going to see two documents. One is an engraving showing Lord Cornwallis, the British general, surrendering to General Washington at Yorktown on October 19th, 1781. And next to it is the letter that George Washington wrote to the Congress about this event. And I think it's always important to see those words that people, our American leaders, wrote and to see their handwriting. So on the next slide, we have our docsteach activity for today's program. Again, that's docsteach.org. And I encourage you to check it out because it features a map from this campaign from 1777. So we'll share this slide again at the end of the program. So at the end of General Washington's presentation, we will have a question and answer session with him. So please write your questions in the YouTube chat box. We have a National Archives staff member who is monitoring it. And let us know where you're watching from today. So this program is brought to you by the National Archives Public Programs and Education staff, the National Archives and the National Archives Foundation. And now it is my great pleasure to introduce to you one of America's greatest leaders, General George Washington. Good day. It is an honor to be here on this day speaking with you all. The campaign season of 1776 dawned with promise. In March, the 11 month siege of Boston ended when the British quit the city retreating to Nova Scotia. The Continental Army then moved south to the city of New York. The one year anniversary of the beginning of the war saw us building redoubts and gun emplacements on Manhattan Island and across the river at Brooklyn Heights. The spring passed in the summer. The Continental Congress declared that these united colonies are and of right ought to be free and independent states. They issued a Declaration of Independence where they addressed the many grievances of the American people and proclaimed to the whole world that we were colonies no longer now United States. The Declaration of Independence came as a validation for more than a year of struggle. It was my fervent hope that it would breathe new life into our military endeavors and additionally help us in the all important contest to win the hearts and minds of my fellow Americans. So it was with a renewed sense of purpose that we dug in and awaited the enemy. We met the British on Long Island and then at Kipps Bay and Harlem Heights each time we were forced to withdraw. morale was not improved when the British began to blow hunting horns when they attacked as though they were engaged in nothing more dangerous than a fox hunt. We were forced to abandon New York and after another encounter at White Plains we crossed over the river and retreated across New Jersey. The Hudson belonged to the British and we retired to the relative safety of Pennsylvania. By early December the promise that had begun 1776 had vanished. With winter fast approaching normally a campaign season would draw to a close. The tradition at least in European theaters of combat was to fight spring, summer, and autumn and then spend winter in camp hoping you don't lose too many men to disease and sickness. In addition to the change in weather another thing that was fast approaching was the end of the men's enlistments. It was a problem that we had faced 12 months before. Congress had authorized enlistments for a period of only one year so the army that we had in 1775 was not the same army that we had in 1776 nor would it be the same in 1777. Short term enlistments were the bane of the army early in the war. You take a man who has just left his farm or shop or his father's house, you spend months teaching him the rudimentary exercise of a soldier. By the time you finally have him knowing his left foot from his right foot and which end of a musket to hold on to, you barely have six or eight months use of him before his enlistment has expired. The enlistments for the Continental Army would expire on January 1st, 1777. If I wanted to make use of the men I had only a few weeks in which to do so. The men were in poor spirits after the retreat from New York. They were cold, they were hungry, and they needed something to bolster their pride. Indeed the public needed something. I knew a victory might well enliven the spirits of the citizenry and bring some who remain neutral in our conflict to the patriot side. A defeat, however, might well spell the end of the Continental Army. I had received intelligence that there was a garrison of Hessian soldiers stationed at Trenton, New Jersey. The Hessians were German speaking mercenaries fighting for the British. I came up with a bold plan to capture their position. I determined to cross the Delaware River on the evening of December 25th, Christmas. Christmas morning dawned bright and cold with the sun shining in an almost cloudless sky. The ground was white frozen snow, no longer powder but snow that had frozen and thawed getting a crust of ice on the top of it. Despite the brightness of the sun it gave little warmth and the cold seemed to seep through clothing and tent canvas alike, chilling the soldiers to the bone. The women of the army had been ordered to prepare three days of food for an impending march. Quartermasters had been ordered to assure that every man had arms and ammunition. Fresh flints were issued for every musket and each man was given 60 rounds of ammunition filling cartridge boxes and pockets. The most coveted of the supplies being issued were new blankets that had just arrived from Philadelphia. A few soldiers lucky enough to get one did not wait until nightfall to wrap it around themselves. By midday the wind had shifted from the west to the northeast. Every farmer and fisherman in the army knew what that meant, a change in weather most likely a brewing storm. By the afternoon the wind picked up it could be felt on the ground and seen in wispy clouds racing by overhead. At four o'clock the drums beat the muster and the men began to assemble along the road. Despite the fact that they were being ordered to march on Christmas day the men were in good spirits. Owing to the impossibility of being in a worse condition than their present one the men always like to be kept moving in the expectation of bettering themselves. I have ever been astounded by the optimism of an American soldier. The men of course didn't know where they were going it was a close kept secret known only to the officers. The plan for the attack was to get into the tree line out of sight before the sun went down and then make our approach to the river under cover of darkness. The main body of the army would cross with me and Maconkey's ferry about ten miles above Trenton. General Ewing would then take a smaller force across just south of the town with orders to hold the bridge over the Assenpink Creek to prevent the enemy from retreat. Colonel Cadwaller's militia would cross at Bristol in order to occupy the Hessian forces garrisoned at Burlington, New Jersey thus keeping them from coming to the aid of the Hessians at Trenton. And finally, General Putnam was to cross at Philadelphia and join the south of Jersey militia. The march was to begin at four o'clock in the afternoon so that everyone would be in position before five o'clock. They would then advance to the river where the boats should be already waiting. The crossing to be completed by midnight the main body would then have to travel ten miles arriving at Trenton just before dawn. It has been my experience that military endeavors rarely go as planned. Some regiments didn't set off until after four o'clock. Some had trouble on the road. Every regiment had men who were sick. Some men had no shoes. Indeed, it was possible to track the progress of our soldiers by the snow tinged with blood from their feet. That men were moving as quickly as they could but a delay of two hours even would mean an attack in broad daylight, spoiling the element of surprise. Though the sky had started out with bright sun and clear skies, heavy dark clouds came in the late afternoon. As the march began, a cold drizzle started to fall which became a driving rain by the time we reached the river. By the time we had the men on the boats and were pushing away from shore, rain, hail, sleet and snow were falling all at once. Before we were even halfway across we were in the grips of a full nor'easter. The other crossings weren't going nearly so well. The Delaware River is tidal up until the falls which are located just by Trenton. So chunks of ice flow down the river over the falls but then as the tide came in, those chunks of ice got pushed back up the river clogging the Delaware below Trenton. The other three crossings weren't able to get across. We had no way of knowing it of course but we were on our own. It took 11 hours to get all the men and equipment across. The men who had reached the New Jersey shore collected wood and cut down fences, built bonfires to try to dry themselves off. I set out a screen of sentries to move away inland from the river with orders to stop and detain any local inhabitant that they might encounter. We did not want anyone observing our landing and sneaking away to warn the enemy. As it was, I despaired we would ever get all the men across. I pulled my cloak around myself and sat down on an old wooden box that had once been a beehive. As the hour stretched on, I despaired of any possibility of surprising the enemy. And I thought about calling off the whole operation. I only decided to press on because I knew if we attempted to cross back over to Pennsylvania we would surely be discovered by the time we got all the men into the boats. While the infantry was all across by two in the morning, it took another hour to get all the artillery over. Colonel Knox brought 18 guns. In most European armies, the standard is two to three field pieces per thousand muskets. We had seven or eight per thousand. The Colonel even had an extra gun crew that he placed with the advanced guard. They carried drag ropes, spikes, and hammers. Their orders were to seize upon the enemy's batteries and turn their own guns around upon them, or barring that spike the cannon so they could not be used by the Hessians. Once ashore, it took another hour to get the men into position to march. The officers all knew their place in the column before we left Pennsylvania, but it took some time to get the men organized in the dark, with the storm still biting down upon us. First in line were the advanced companies ordered to operate independently advancing as quickly as possible to within two or three miles of the town and there to put up roadblocks to keep anyone from going in or out of Trenton. Next came Adam Stevens, Virginia Regiment, whose orders were to attack the enemy's sentries and pickets. Behind them came the rest of the army, divided into two columns under Generals Sullivan and Green. They would march for a time together and then split with Sullivan's army advancing along the river on the right and Green attacking the town from the north. Of course, at that point we still thought we had men attacking from the south and east since we had no way of knowing that the other crossings had failed. By the original plan of attack, we were supposed to be in position for the attack by five o'clock a.m. But it was four o'clock when we began our 10 mile march. The road away from the river was to the northeast straight into the storm. Men who had dried off by the bonfires were soon soaked again as they made their way up a rocky hill for a mile and a half to an elevation of about 200 feet above the Delaware. As we got to the top of the plateau, we reached Bear Tavern Road where we turned to the south. The rain and the sleet had turned the road into a solid sheet of ice, but at least it was level. We made better time. That is until we came to Jacobs Creek, small stream that had cut a deep ravine into the New Jersey countryside. Stretcher was enough for the men to march down, but there was no way that the cannon could be hauled down there by horse. A six pound cannon with side boxes and ammunition and trail boxes for equipment weighs more than a ton. It would quickly kill any horse that tried to pull it downhill. Instead, we had to unhitch the horses, pull out the drag ropes, using trees as mooring posts to level the guns, lower them down slowly into the bottom of the ravine, more than 100 feet below our starting point. Once up the other side of the ravine, the ground gradually sloped uphill till we came to an open plateau. The ground was level, but having left the tree cover behind, we were completely exposed to the northeaster. At least two soldiers simply laid down at the side of the road and froze to death. Gradually, the plateau gave way to a long smooth down slope. We once again had to make adjustments for the artillery, so this time we did not have to unlimber the horses. We had to have men on the drag ropes to act as brakes, keeping the artillery carriages from running over the four animals. By the time we caught up to the advanced parties two miles outside the town, it was 7.30 in the morning and the sun was up the week, could see no hint of it through the storm. I gathered my officers around me in a brief council of war and it was discovered that many of the men's arms were wet. Indeed, not only were their muskets wet, but many of the cartridge boxes had been exposed as well. It was very likely that a goodly number of our weapons would be unable to fire. I was with General Green's division on the north side of the town. Not knowing how many of our weapons would fire, I gave the order to advance and charge. As we broke through the treeline, running through the falling snow, we saw a Hessian officer emerge from the door of a Cooper shop. I saw him look right and left down our lines as though he were trying to decide who we were. Doubtlessly, he thought we were a returning patrol at first. He was dissuaded of this notion when one of the men fired at him, narrowly missing. He dashed into the shop and a moment later, a company of Hessians poured out, formed up and began to fire. Our men fell into ranks and gave them three ragged volleys, driving them back. Moments later, I heard cannon fire coming from the west side of town. General Sullivan had engaged on the right flank despite the difficulties and delays we had still managed to surprise the enemy. And it was not the meticulous planning, friends, no. It was the storm. It was the tenacity of the American spirit and the endurance of the American soldier that allowed us to push the Hessians back to the center of town. There's a myth, sometimes perpetuated among Americans regarding the Battle of Trenton, that the Hessians were suffering from the barrel fever, having been drunk in Christmas celebrations the night before. As the battle progressed, we saw no evidence of that. The enemy, particularly those at the outpost, formed up in good order and fought well, even while retreating, pausing at each building or intersection to return fire. Regiments that formed up in the center of town had evidently been sleeping in uniform with weapons and ammunition close at hand.  Even the horses used to move their artillery had been stabled with harnesses on, ready for an attack. We had the element of surprise and superior numbers. And the effect of our artillery that took that extra hour to come over, so many guns brought to bear upon the enemy could not be overstated. In addition, General Knox had stationed a battery of halitzers on the Pennsylvania shore, which joined in and bombarded the center of the city when they heard our attack across the river. In 90 minutes, we had taken to town along with almost a thousand enemy captured. Around noon, we began to move back to the boats with our prisoners and captured supplies. Among the spoils were 40 hogs heads of rum, which I ordered to be destroyed. However, some of the men availed themselves of this libation, perhaps thinking that drinking it was the most sensible means of destroying it. More men fell out of the boats on the way back than had on the way over. News of the victory at Trenton spread. This had two effects. Among Americans, it was proof that the Continental Army could stand up against a European foe and win. The enemy, it was seen as proof that the Americans could strike anywhere. They began to look out from their winter quarters into the snow, thinking that Washington and his rebels might appear at any moment. New Jersey was like an anthill that had been kicked. Reunited with the rest of the army, I desperately wish to recross the Delaware and press our advantage, but there was one serious problem that faced us, just as it had one year before. The men's enlistments were about to expire. In a few short days, there would be no army. We finally managed to get the whole army back to New Jersey and three days after the Battle of Trenton, we returned to the town, but at that point, there are only two days left in the men's enlistments, not enough time. So I made my appeal, my brave fellows, you have done all that I have asked you to do and more than could be reasonably expected, but your country is at stake, the wives, your houses, and all that you hold dear. You have worn yourself out with fatigues and hardships, but we know not how to spare you. If you will consent to stay only one month longer, you will render service to the cause of liberty and to your country, which you probably never can do under any other circumstances. The present is emphatically the crisis, which is to decide our destiny. I'm not sure if it was the speech or the $10 bounty that was offered, but I was able to induce almost 1,400 men from the New England brigades to stay. Added to our other troops from Virginia and Pennsylvania, we had a little over 3,000 men. British General Charles Cornwallis attacked our position on January 2nd, but we were forced to retreat through the town of Trenton. We established a defensive position on the Assamping Creek. Cornwallis assaulted our position three times and was repulsed three times. This second battle of Trenton had begun quite late in the day. Cornwallis retired, thinking to finish us the following day. However, we withdrew and counter-marched around Cornwallis' camp that night to attack the British Reserve at Princeton. We left 500 men behind to keep the fires burning and to begin to work with picks and shovels so the British would hear and think we were digging in. Before dawn, those men abandoned their position and joined the main army. By the time we arrived in Princeton, once again, later than was hoped, two British regiments were already on the move, having likely been ordered to join Cornwallis. Colonel Cadwallidher encountered them first, but he had a hard time getting his militia into a battle line formation as they had not had adequate training. When I arrived, they were beginning to flee, so I shouted, parade with us, my brave fellows. There is but a handful of the enemy and we shall have them directly. I formed them into a battle line and we began to advance. I ordered them not to fire until I gave the signal and I rode in front of them. When they were 30 yards away from the enemy, I turned around on my horse, faced them and yelled, halt, and then fire, and they did. And so did the British. I later found out that one of my officers had pulled his hat down over his eyes. He did not want to see me be killed. Yet when the smoke cleared, there I was, unharmed and waving the men forward. We advanced forward into the town and the British continued to fall back. Some few took refuge in Nassau Hall, which is the main building of the College of New Jersey. One of my future aides, who some of you may have heard of, a young artillery captain from New York named Alexander Hamilton brought up three guns. I suspect some of Captain Hamilton's enthusiasm was because he had once applied to the college and been turned down. On January 3rd, 1777, he indeed got into the College of New Jersey. Shortly afterwards, a white flag was timidly waved out a window and the day was ours. The battles of Trenton and Princeton were small victories, but important. In the six months since our declaration of independence, the American people had begun to lose faith. Many were of the opinion that it was best to surrender and be done with his hopeless war. This is when Thomas Payne wrote the first of his essays titled, The American Crisis, in which he said, these are the times that try men's souls. The summer soldier and Sunshine Patriot will in this crisis shrink from the service of their country. But he that stands by it now, deserves the love and thanks of man and woman. Tyranny, like hell, is not easily conquered. Yet we have this consolation with us, that the heart of the conflict, the more glorious, the triumph. The victories in New Jersey made people believe in that triumph. Christmas and New Year have ever been a season of hope and renewal. Certainly that was the case in 1776, when the endurance and tenacity of the American soldiers saved our new nation. Well, thank you, General Washington, for sharing that stirring account of those 10 days that made such a difference in our history. So we do have somebody who's jumped on from Vista, California, and thank you so much for joining us, Brighton, early out there. So we do have a few questions for you. Certainly the, and I had mentioned it in my introduction, the very famous painting showing you crossing the Delaware River in the midst of that storm.  Almost everyone was standing for the crossing. We were in, well, we were in any boat we could get our hands on. The New Jersey and Pennsylvania militias had been collecting boats. When we, from the time that we retreated, they had collected every boat so that there would be none that the British could use to get to us. We brought them all over the Pennsylvania shore, hid them in little coves and streams. And so, but the bulk of the troop transports that we used were actually these Durham boats, which were a long flat bottom, they were a variety of sizes, but long flat bottom boats that were actually intended to transport iron ingots from the foundry that had, where the name comes from. And iron ingots, anyone who's ever seen an iron ingot knows that it is somewhat heavy, not as tall as a man and doesn't move around much. So it doesn't require a seat. So those are the boats that we were being transported in. And so there were no seats to speak of in them. And so the men mostly stood. Add to that the fact that it was raining and hailing. So the bottom of every boat had several inches of ice water. We would not have been in good shape had we been sitting in ice water on the way over. No, that's for sure. And you were able to transport the horses in those as well. We generally, there were also fairies that we took on the, those are intended to transport horses and carriages and things of that nature. So we tried to use those for the horses for the office sources for the artillery pieces. So those were what we used to transport the horses over. As it was, there were perhaps a third of the horses that were lamed by the time they got over just from standing in the ice and wind for that period of time crossing the river. Okay, all right. And then another question kind of going along with horses.  Is that in fact true? No, not exactly. It's been exaggerated as many of these stories can be over time. It was on the March in from the shore on the Jersey side. It was just after, it was actually, I believe it was up the way the ravine after Jacobs Creek, I mentioned that large gully that we had to traverse. And my horse, the rear legs skidded out from under the horse and the horse was in danger of falling down. So I didn't actually, I was on the horse. Can't pick up the horse, the one is on. But what I did is wrap my fingers in the horse's mane and lift the horse's head, essentially shifting the entire horse underneath me to shift the weight so that he could get purchase on his back legs and get stopped skidding on the ice. So I did move the horse by lifting the front, the lifting the head of the horse pulls the chest forward and then shifts the weight to the back so that the horse could get purchase with his back legs. Because that, and some people have accused me of being cruel to the horse by pulling on the mane and lifting the head up, not as cruel as landing on top of the horse on top of icy rocky ground. And that probably that knowledge of how to do that and how to control the horse in that situation comes from your many long years and knowledge and understanding of working with horses and riding. I'm sure that that was something you did from the time you were a very small child. Very much so. Yeah. The most earliest memories are of riding. Riding? Yeah, and that obviously that came in as time went on. So during this campaign, what do you think was the most significant factor? If you could choose one that allowed for these multiple victories in a time that was so needed? I feel, well, Colonel Raul, who was the Hessian commander at Trenton, made no effort to fortify the town. Had we had to charge against earthenworks, for example, we would have had a more difficult time. So it was, their defense was literally men forming up like they might in an open field. They used buildings for cover, but there were no earthworks. There was no palisade or wall thrown up around the settlement. And we also came in, with General Green's division, we came in on the north side, which is high ground. So from my position, whereas Colonel Raul was at the low ground in the center of town, I could see where all the German troops were moving as they were moving from point to point. So we came in with the high ground, the storm allowing us to still have surprise, even though we were late getting into position, those were, I think, the most valuable factors there. Okay, well, that's great to know that. We got somebody from Columbia, Maryland saying hello. And then let's see, you did talk about people falling in during the crossing. You said that more people actually fell in going back because of the rum and going over any percentages, number of people, and what happened to those people? More than one, more than one. On the way over, so two very different crossings, obviously one during broad daylight with the storm having abated, that was our return crossing, but our crossing over was done in terrible, terrible conditions. Wendy, and again, these boats, everyone is standing up in these boats and they have sides on them, which are maybe 24 inches. So a man standing near that, if somebody jostles so many loses his balance, he falls in. We did not lose anyone on the crossing, but men did fall in and had to be dragged out by their compatriots. Please keep in mind, most of these men cannot swim. And even if they were able to swim in the cold water, they would not have lasted long. So they would, they got out and got to the bonfires as quickly as possible. We did not lose anyone, but yes, men did fall in. When they fell in because of drunkenness on the way back that was their own fault. Yeah, they had to figure out that on their own. Exactly. Well, so thank you so much. And we do have one more question for you. And this is, so what advice from back in those days, 17, you know, 76, 77, or even looking, you know, forwards with your life all the way to 1799, what advice would you give to young people today? Thinking of the lessons learned in the, the forage war, as it was called the New Jersey campaign, every nation at every time will face crises. Every generation will have problems to overcome. What won those battles in the jerseys in 17 and 76, 17 and 77 was the ability to endure the hardships that we were suffering and the tenacity and the will to continue even when it was difficult. That is the advice that I would give to young people of any generation, whether they face battle against German mercenaries or any other number of problems that might come upon a nation and a people. Yeah, that quality of stick-to-itiveness I think is, you're right, it's so important for young people, for everybody, because people do face challenges. There's no question about it throughout their lives. So this has been a wonderful discussion, talk with you and learning so much about those important 10 days in our history that made such a difference. There were no doubt that it was a turning point for us, for the United States in so many ways. So thank you for joining us and I wish you and the best of holidays. Madam Petters, it's been my honor. Thank you. So as I promised, we, here is the Doc's Teach slide again and I encourage you to check that out. And again, it's docsteachdocsteach.org, you can see the map up there. And then finally, the program in January is with Fannie Lou Hamer and it is on January 20th at 11 a.m. with Sheila Arnold and I hope that you will consider joining us for that. And thank you for joining us today and I wish everyone happy holidays and a terrific 2022.