Let me share some thoughts this morning with you about the church. We love most special thanks to To a couple of people whom I've read from read their most recent books. Mr. Dr. Gil Rendell and The former Dean of Perkins School of Theology Bill Lawrence Over the last several months. I've been thinking about churches of which I've been apart from the time in which I was baptized as an infant I've shared similar pieces of this story with you before about my own call to ordain ministry But I think there's a closure to this that it decided I needed to have at least with myself Not only is a closure about my own life story or faith story. It is also a Time of lament and challenge. I think for the church So I began to think about all those churches the first church of which I have any recognition of was actually the church in which I was baptized is the church where they extend them the key family went my grandmother my aunts my uncles my cousin my dad and I and my little brother from the time I could remember Set on the back pew all my mother saying in the choir. I was baptized in that church Sort of grew up partially in that church. I don't remember my baptism, but we'll I'll remember it in a few moments as you will yours That church closed 20 years ago That church closed 20 plus years ago But the time is in the fourth grade now family having moved to another part of the city of Fort Worth Joined a church in the neighborhood. It's where I spent my childhood my adolescence It was the place in which I really began to hear the call to ordain ministry in terms of my own life That church closed in January of this year following that In my first church job having transferred my membership To a church in the city of Austin in fact the first time at this church of Austin returning home after graduation from the University of Texas I began working at a church called Richland Hills. It was my first church job The best part of that was not only the people I got to work with in the congregation Really the best part of it's the place in which I met Joan Craig McKee and we were married while I was on the staff there That church was about three miles from the church in which I spent 15 years of my life while I was at the first Young Methodist Church of Hearst the Richland Hills church Actually changed its names did some other things and it worships less than half of the number of people who were there in 1973 as Privileged to be appointed to what was considered a fairly large church in those days And that is a church worshiping for hundred people and those were large churches before there were these large Mega churches that came into being that provide a host of ministries That church was on the same side of town in which I grew up at the first of this year It became a part of another charge so that the formerly Second largest church in the central Texas conference along with the Medebrook church where I served was the 10th largest church now They are on a two-point charge As I've reflected about that I've begun to ask questions about how in the world did that happen What is common about all of this is that they had good pastors good people And for churches of their own size They were much better than average But the ground shifted The ground shifted for another reason number of reasons. They're not necessarily the shifting grounds that we have today Of which I'll speak later But it really was the ground shifting due to the cultural quake. It's like a seismic shift in the Christian landscape They had as much to do with what was going on in the world as it did in terms of how people lived out their faith And really what I'd say to us that Gil Rendall says so clearly is is that there's a difference between a public mission and a private mission We all know what the public mission of the United Methodist Church is don't we? to make disciples of For the and while that was not the mission statement then necessarily we were not even dealing in mission statements in the 1960s really It's given that both clergy and laity like we're doing and are doing good work It was happening then it's happening now But now over the years it may have been done with a lack of awareness of all those shifts that were taking place in the culture and in society and in education and in politics And looking back we now know that it's true We could begin to talk about the timeline and when that happened and what happened when we couldn't have seen it coming But it came and it is still here And it's past time Forced to at least acknowledge those shifts Because they have happened they are happening they will happen and we sometimes get confused about our commitment to the Christian faith and The way we live out the faith and the manners in which we do disciple making or even do church We put both of them in concrete put our feet both of them in concrete in both camps In terms of theologically in terms of the way in which we do ministry While some of us at this time We're baby boomers. It was a time in which we thought everybody went to church on Sunday morning In fact in a couple of conversations I've had with clergy her about my age They said, you know, we live in a time in which our grandchildren were no longer go to church every Sunday because everything that was happening around them and for them Competing with all the other things that take place on Sunday morning It is not as if we are the only game in town not to say that church is a game. I am to say though We don't have Sunday by ourselves anymore Now we can decry that we can talk about it. We can rail against it. We can say why do people do that? That's not the place. It's then that is a way of living in yesteryear It is time for us to live not only in today, but into a future And so how do we begin to then shift our feet as the ground shifts in such a way that we are as concerned about how do we reach People for Jesus Christ on Monday morning Monday night Wednesday night Not in terms of the old prayer meeting kind of thing I'm saying but at different times the week different points of way people engage so that people begin to have a living relationship with Jesus Christ So that's what we need to be tied to And we do not need to be tied or place our feet in the concrete It was good enough 30 years ago by golly. It's good enough today Because it's not good enough and it began to think about the decline of the Christian witness in this country Yes Even in what at one time was called the capital the Bible Belt Dallas, Texas Where people are allegedly more religious in Dallas than anywhere else place in the United States? friends It's happened here It's happening today Response too often Has been this A church consultants sort of some church consultants majored in this well-intentioned But still failing Too often we've said let's do what we do better Let's just improve and you can list off the number of things all of which need improvement But it may not do what God is calling us to do So let's improve our music. Let's improve our worship. Let's improve our preaching. Let's retool Sunday school Let's be a thousand families at Thanksgiving instead of 500 this year Let's call all the inactive members of the phone and by the way And while it may have made some changes it is not sufficient for ministry in today's world Gil rendels drawn upon the work of Robert Quinn as professor of organizational behavior and he writes Gil does of religious institutions I really am sort of tired of us referring to the church as an institution Can I just get an amen about that? Because I this is not in the script This is what This is when I can be at my best Which is unfiltered So so so let's be honest about this In terms of a public and private mission We prefer a private mission Which is taking care of all of us Making all of you laity.  I'm sending clergy to you To be the pastor of your church To make you Uncomfortable at times. I'm sending persons to make you uncomfortable for this reason because frankly The witness to Jesus Christ calls us to all rethink and then reclaim what it means to follow this Jesus the Christ It's to engage in the public mission to make the cycles of Jesus Christ for the transformation the world And you know, I've pastored people to and you and lay persons. You've had clergy that Just didn't get it. Well, let's be honest about it It's not worth picking up anybody's marbles and walking out on each other about anything And the reason I say that is because I'll never forget this a Good friend who's the who's the late person of a large church non-methodist, but one of the mainliners Was sort of visiting with some of his fellow church members about something that not everybody agreed with and I'll never forget I'm saying Mike it's how I've told my friends This is my church family. This is really about my faith family And I don't walk out on my wife when we have a disagreement And I don't walk out on my faith family because we don't all agree Making disciples of Jesus Christ is hard work. It's harder now than it's ever been We should not expect the culture to be kind to us It was not kind to Paul as Tom Bickerton said so well last night It won't be kind for us, but you will not suffer quite like that I can promise you that you will not be confined to a jail cell because it you will not have changed You'll have plenty of food. You'll have plenty of water You'll get to sleep tonight or any other night in your own bed It is a lot easier than it was 2,000 years ago. Let's quit talking about it is so hard It is not that it's hard It may be that we do not mind our own deep spiritual lives or the life of congregation to do the work That is God is calling us to do and again, I say it's not in the long run For all of us to be happy about everything that happens The goal is is for the interior mission for us to be joyful to be with great joy About giving to live the faith and the communities in which we live So the claim of the public mission is that we actually believe that God can change people to change the world and That gets our passions moving But the private mission is what we have sometimes need term near terms which is rewarded in unusual ways The private mission focuses on ourselves both clergy and laity like But the public mission focuses on our charge our purpose our calling as Christians The challenge for clergy has been in Today's world is how can you be open and engaged with the mission field? In fact, it's the way in which the cabinet and I make appointments Contrary to what some of you may think There's a great deal of prayer and purpose that goes on in the making of appointments As slow as the process seems to be to you Know that the process sometimes seems slow to you because We want to do good faithful work and we begin with God's purpose We begin to take stock of the mission field And our superintendents know the mission field so well of their churches that we can call on them and they can tell us quickly So it's God's purpose and then the mission field and then we began to look at the church as a steward of the United Methodist witness In particular community Which means it is a mission statement to live out the public mission of the United Methodist Church the North Texas Conference in a way that sometimes Can make us a little uncomfortable and contrary to way appointments were made 40 years ago It's not that the last person we think of it's the pastor It is as a her is the biggest concern has to do with God's purpose the mission field the church and then the pastor Frankly, we what we do is in terms of the appointment system is really seek to somehow place deploy clergy Not just to grow church, but to deepen their spirituality It is really a call To do that which will provide the most joy Now let's be honest what a mission field is It is a church. It is a community that's full of people who have no relationship with Jesus Christ It's not just a one-time episode or experience that you do in that in that mission field It's an ongoing vital relationship. I shall never forget about how I learned about this first hand I was preaching in Rochester, New York a Number of years ago At the largest church in Rochester 8 or 900 people worship their own Sunday morning a big Gothic building. It's in Rochester, New York I've said to the pastor gosh the music is great in this place Bob Hill said to me Mike if you don't have great music in Rochester, New York with the Eastman School of Music here You need to just fold up your tent and leave, you know And then I said to him I know how hard this must be an upstate, New York Or you know with declining this this plant closing that happening that happened. He said that is an excuse For not engaging in the mission because I can promise you Mike There is many children and school today in this community Then there were 40 years ago when Eastman Kodak and Xerox were getting started And we can find a number of ways to no longer engage But I promise you if there's just a school in your community there are people there And I can tell you they don't all go to go to church And it's not about going to church is about our Bible relationship You know our churches need to look like our communities And one of the common things that happened to some a couple of the churches I named is the community began to shift and change color in 1992 only 6% of Americans Rejected any form of religious affiliation today. It's 25% It's even higher for millennials Church attendance is declined twice as much listen to this twice as much among Anglos without a college degree than any other group Friend here's where it is We are a middle income upper middle income group with a few exceptions and Unlike our founder John Wesley who believed that he had to become more vile His words to even meet reach the mission field. We don't know how to have conversations other than providing a meal Or some act of mercy When what we need to do is engage in a very significant conversation About what a relationship with Jesus Christ can do I will go ahead and confess my own failure about this Because we simply don't know how to do ministry within the large numbers of our mission field and to be honest We need to find people who do and we need to trust them to teach us more than we know the mission field is the place in which We must do ministry differently I begin to think about some of the things that are happening over the North Texas conference There are many more than I'm able to list here today One of them is part confessional again for me Shortly in my time here We began to talk about some experiments in some places The first one I remember is white rock I've talked publicly about this even with the council of bishops when they met here in May a year ago or two years ago I Introduced Mitchell Boone and introducing Mitchell Boone. I did say to the council of bishops I said I must confess to you that What has happened at white rock? I didn't believe could happen. I Thought with its endowment we should close We should take the money start a new church somewhere else. I Think that that was the conventional wisdom enough people I was worried about spending the down the endowment which I consider ways in which we can do ministry and somebody said let's just try it I Won't detail everything that's happened. That's a conversation you can have with Mitchell Boone But I would say to you and I think Mitchell would too you cannot recreate what's happened in one place You just have to be imaginative to do it in the place where you serve So when you talk to somebody who's made something happen, don't go copy it think okay What questions do we need to be asking about our own mission for you? And as I introduced Mitchell to council of bishops, I said I I'm gonna make a confession in front of Mitchell I never said this to him before I said I never thought this was this would work. I Thought it would not work And so I'm saying before all my colleagues that and happened very often in that group. I want to tell you I Want to confess that I was wrong They all laughed I Think they admired it more for this candor to which then when Mitchell Boone spoke he did said don't worry Bishop I didn't sleep for two years And the story of white rock of what was happening and what is now happening And this is not an indictment on anybody or anything that took place before then is just nothing short of miraculous It's younger It's more diverse. It has some other things it's doing. It's taken over a closed church, Owenwood to start a new ministry there frankly friends think creatively Think imagine Lee think boldly For God's sakes remember that there are people there who do not know Jesus Christ and Meet them and invite them and be patient with them There's several other things that are happening in terms that Lakewood started messy church That's difficult when you think you want to keep the walls clean the carpet clean and everything else I don't really know everything that's messy church I know that our grandson went to messy church in the Pacific Palisades a few times. I know it's for children I knew it's a new way to engage with children and it is a way to engage with young families who may think that they're too busy The first year I met this church of Allen is engaged in a significant building project Many of you have done those You know how to do them But there are people who are always on that site so that every Friday The first United Methodist Church of Allen furnishes a meal for anybody who's working on the site that day Now you can imagine over the course of a time of a building project that would change from week to week to week Not only do they share a meal they share a devotional and prayer and the connections They've made with some workers who come and then don't show back up because they've finished their work there Some of them have said I've decided to go back To the church And you know what? It's not just about getting members. It's really not It's just about reacquaining people with Christ occasionally first Paris is engaged in a challenging piece related with City Square in Dallas to call City Square in Paris to address the Continued the continued poverty issues that are related and related in Lamar County with persons who living in poverty by doing some creative things including housing It happens it can happen to you Or you can make it happen and I pray that you do Lastly, I'm not lastly. I also want to name the elephant in the room They're actually two Two elephants together in a room this size could be dangerous It could make us all uncomfortable it will But I'm reminded that Paul frequently told the church. Let's be mature about how we live together You may not know this some of you But the general conference in the item at this church mate met in St. Lucer three or four days in February It's the way I celebrated my birthday this year Actually, it was about the day before the bishops arrived for the council bishops meeting beforehand But what I want to say to you it was the purpose is right some clarity But it did not I know for you some of you in the room and you think it did From my position and what I get in terms of different things and the conversations I've had with people around the conference The clarity it brought has been painful Some people say that the traditional plan passed But to be honest as it was presented some of it passed some of it didn't Some of it was ruled in unconstitutional some people say Well, nothing changed Well, nothing changed about our position. There's no doubt about that related to weddings of same-gendered persons in our churches Or LGBT clergy? But there is something that passed and was rule constitutional And that is the way in which persons are held accountable specifically clergy if they were to do a same-gendered wedding There are any number of views on this point. I'm not talking about that right now But I want you to hear this This is what's changed significantly Any complaint that's received on any of our clergy is processed Nowhere in the discipline until this general conference Was the sentence mandated So for instance a person could commit adultery who could reach a gesture resolution in such a way but this time If to if a clergy marries the same-gendered couple And the just resolution is not met and a trial is had immediately that person is placed Placed on leave for a year without salary or benefits We don't do that for a clergy who steals money We don't do that for adultery necessarily. I'm not saying we wouldn't what I'm saying It's the only place where a punishment is mandated There's a lack of fairness about that and Those who even have what I would call very very very conservative opinions about same-gendered marriage They even acknowledge that's a bridge too far friends It is the only infraction on the book of discipline that carries a prescribed penalty and to be honest That ought to put in question Even our polity Frankly the backlash from that general conference is Becoming quite evident across the country Some are asking what will happen Some have asked in this annual conference who are unaware of everything that was happening late the what happened to the church but the polity and I think many of my colleagues share this many pastors with whom I've taught it's simply untenable It's not about this. It's simply untenable For all the beauty and Bishop Bickerton talked about this in a different way yesterday for all the beauty and hope of a global church It can't work the way we're structured now So let me share with you what happens We really don't you know if we can sort of not talk about this stuff and everything you'll feel better Until five years from now. You won't feel so good And you'll wonder why didn't Bishop McKee tell us this or why didn't our pastor tell us this which is a question I asked many times So here's the polity question There are four Areas of the church the central conferences in Africa The central conferences on the continent of Europe the central conferences in the Philippines the central conferences in the I mean Excuse me the jurisdictional conferences the United States. So every several central conference, except we don't have a central conference here We have jurisdictional conferences. I'm getting messed up here So every central conference in the Philippines and Europe and on the continent of Africa write a discipline for their context You know this book that keeps getting thicker about who we are or what we're to do I Say the best thing we could do is make it a book of marching orders to reach people for Jesus Christ and cut it out about four-fifths of that book And let me be clear. This is not about human sexuality It is about the way in the context in which we deliver that frankly So every four years we together and all the central conferences across the country good faithful men and women clergy laity are voting on how we credential ministers in the United States and they return to their own Context and decide that for themselves look I Think we can be united met this and have different ways in which we govern in our polity from context to context in the United States as opposed to Europe or Africa or the Philippines But this is one thing that this book the discipline one side doesn't fit all But there is a book that does and that book It's called the Bible With which you know methodists use our tradition our experience and our reason to interpret To discern how it is that the spirit is moving in our midst today Friends people ask me what should we do? What are we going to do? And to be honest I think in time Maybe in sooner time than we know That the spirit will be moving This is what I do know I do think United Methodism Will have a new reality. I Don't know what it will be We have to wait for the legislative body decide that Many of you think we had we as bishops have more power than you can know. Oh friends trust me that's simply Been a myth that you have believed or that you've been taught or you would think would make sense But this is what I know There's a moving of the spirit of in Methodism But it's not just about human sexuality. It's not just about polity There's a movement and acknowledgement for what I think is an even bigger issue and Every time I come to annual conference. I begin to think wish we could make this room Look like the mission field in North Texas And what I'm and what I mean is race Racism is still very much alive Even in the church, so this is my charge to you about that Could you just acknowledge it? I mean awareness is the beginning point of learning Could you begin to counter it in such a way in which you are able to say to a good friend of yours who says just one racist thing too much Hey, we need to talk when your pastor goes ahead and speaks about race Could it finally be the time in which I don't get an email complaining about your pastor For God's sakes I Get more emails about your pastor talking about race or Immigration or children housed on the border when they talk about it I get more emails about that than I do on human sexuality I'm telling you this is a big issue and whatever your policy or your thoughts about the policy or our politics or anything else for God's sakes Could a child of God who's seven years old at least be in a place that would be safe When they came across the border for God's sakes It matters not to me whether you are a Republican or a Democrat or your view on immigration But let's at least pay attention to children so that they don't die in the hands of our government So this is what I hope to see at some point in my lifetime. I Hope to see this is a vision for the church That when we gather as an annual conference 10 years from now that we will have discovered that we have learned how to meet welcome incorporate train enough Spanish-speaking pastors in order to reach 42% of the population of Dallas which speaks Spanish we have We one point six percent of our clergy or Spanish speaking or Hispanics and they do good work We need more people to do that work It means that sometimes we realize that our policy will be done a little bit different just in order to credential persons to do that work I look for a time in which you as clergy and you as laity live in such a way and Which you think about what it means to be a follower of Jesus Christ and you realize that it comes from our Wesleyan tradition Which is so strong and the reason I'm a method is simply because it's a theological one It's not because my parents went there. It's not because my grandparents went there It's because there's this place about grace in our lives that somehow when you and I didn't know it and When we turn the corner of our life Our God and loving kindness meets us there It is about the grace that which always surrounds us It's about the grace which then greets us It's about the grace which transforms us But it's not just about that one-time experience and it has to do with our own our own Response it's that we continue to reform by that grace It's not a one-time and it's fine It's that we grow West those who are getting ordained you expect to be made more perfect in this life. It is a good vision to have So for the church we realized that what it means is I would love to see a church in which We were all growing in that grace that every moment of our waking lives We think about how We really are growing in the depth of the faith. I see a church In which we meet people where they are Despite their income level despite their race despite their culture and we share with them the good news Maybe so God bless you


Exploring chiropractic, episode 34, primary spine care with Dr. Donald Murphy. Does chiropractic training prepare students to be primary care practitioners similar to the family medicine doctor? The answer is emphatically and absolutely not even close. Hey guys, I want to tell you about Backblaze.com. Backblaze is unlimited data backup for your Mac or PC for just $5 a month. It's unlimited files, unlimited file size, unlimited speed. And get this, it will back up all of your attached external drives. That's right, so not just the stuff on your computer, but you've got your old photos, your old movies. And you can back up everything that's attached to your computer. Check it out exploringchiropractic.com slash Backblaze. Welcome back to Exploring Chiropractic, the only student chiropractic podcast. I'm Dr. Nathan Cashin. My guest this episode is Clinical Director of the Rhode Island Spine Center and the Director of Primary Spine Care Services for the Care New England Health System. He also serves as clinical assistant professor in the Department of Family Medicine at the Alpert Medical School of Brown University, the professor at Southern California University of Health Sciences, and adjunct associate professor in the Department of Research at New York Chiropractic College. You may have heard of the clinical reasoning in spine pain textbooks which he has authored, and I'm very excited to bring him on the podcast to discuss his background in chiropractic, the concept of primary spine care, and more details about the CRISP protocols. Dr. Murphy also answers some listener questions. I hope you enjoy this interview with Dr. Donald Murphy. Dr. Murphy, thank you for joining me on Exploring Chiropractic. It's a pleasure to have you on. Thank you for having me. I was looking through your CV. One of your previous interns or preceptors, I'm not sure what you call it, Dr. Brendan McCann recommended I take a look at your CV, and I haven't been able to get through the whole thing. It's quite a volume, but the one thing that stood out to me is you started in acting, is that right? Yes, I was a theater major before I switched majors to pre-chiropractic. So what took you from acting to chiropractic? Well, my first two years of college, I was again a theater major. I was studying acting, and that's what I wanted my career to be. But as I was going along, I decided that I loved acting, but I didn't have the level of passion that is required to live the kind of life that an actor has to live while they're struggling to make it. So I wanted something more steady and more stable, and so I actually took a year and a half off after my first two years to find myself before deciding to go back to school and become a chiropractor. Did that time often involve any world travel, any type of voyages that helped you find yourself, or was it just taking a break? Well, not world travel, but country travel. I grew up in New York, and I was living in New York, and so I moved to California, which in the 70s, early 80s, that's what one did when one was finding oneself, moved to California. So I lived in Newport Beach, California for almost a year, because kind of working and living, and again, I didn't go out there to set out to find myself, but that's what ended up happening. I got it. So from there, how did you get into chiropractic? Did you have a miracle chiropractic story, or was it just something you were aware of? Well, I came back to New York after living in California, and I had a friend who was just getting ready to start a New York chiropractic college. So he would talk to me about chiropractic, and it sounded really interesting. I was always interested in health and fitness, and so I was naturally intrigued to anything related to health, but also something natural and focused on an act of lifestyle and that kind of thing, and so I became intrigued, and then I developed a shoulder problem, and so my friend encouraged me to see a chiropractor about it, and the chiropractor was able to help me, so I said, okay, this is something that I want to go into. Interesting. The way you describe that, it almost sounds like kind of a spontaneous decision. How much time did you deliberate on going into chiropractic? Well, we're going back a couple of years, so I'm taxing my memory a little bit, but I don't think it took very long. Once I decided, okay, I want to have a direction, and I started talking to this friend and had the chiropractic experience, it was pretty quick that this is the direction I wanted to go in. I find that interesting because recently, I heard a very similar story. I don't know if you were familiar with Dr. Lester Lamb, who's well known out here at Western States, and he just passed away, and so at his memorial they were playing a video, and he told a very similar story. He was in the military, the Peace Corps, he came back, didn't know what to do with himself, and a friend was enrolling in chiropractic school, and he kind of said, okay, I'll do that too, which seems very different to, say, my experience, where I probably deliberated for about 10 years before I actually started school. Yeah. So you went to New York Chiropractic College. Yes. And after that, I understand you did some postgraduate training. Yes. I first did the postgraduate training in certified chiropractic sports physician. I got that certification, which was, I think, 100 hours or something like that. And then I started the neurology diplomate program, and that was a three-year process, and I earned my diplomate in chiropractic neurology, quote-unquote. And during that time I became very passionate about neurology and neurodiagnosis, and so I supplemented my diplomate studies by making myself a pest to the attending physicians at the local teaching hospital in the neurology department until they let me come and do rotations with the neurology residents. And so I spent over 200 hours doing rotations with the residents in the various clinics, the neurology clinics, and really getting a great education in neurodiagnosis. At this time you had already graduated. Were you in practice? Yep, I was practicing at the same time, so it was a very time-consuming process. Yeah, and I find it interesting that you were able to, after some pestering, as you say, convince them. I think a lot of students really would love to have an opportunity like that, but I certainly have tried a couple of times and just gotten so much pushback or disinterest that it was hard to keep trying. What led to that persistence? Well, I think that what was very helpful to me was that I showed myself to not only be persistent to these folks in the neurology department, but also passionate about learning and sincere about learning. And so this was a teaching hospital, so the people who are involved, the attending physicians there are teachers, and as a teacher myself, and you can imagine because you said you taught yourself as well, teachers love to teach. And so there's nothing that a teacher loves more than to encounter somebody who's passionate about learning what they have to teach. And so I inadvertently appealed to that aspect of their nature and I think that was my biggest asset. What did you do other than doing the Diplomat program in neurology? And was that, by the way, was that through Carrick at the time or was this a different program? Nope. It was run by a guy named Dr. Joseph Ferrissie who I think still has a course running and it was through the New York Heart Practical College. Oh, interesting. I hadn't heard of that one. Okay. So outside of the Diplomat program, what types of things did you do to educate yourself to prove your devotion and passion for learning to allow you to get into these teaching hospitals? Well, in addition to and separate from that process of taking the neurology course and doing the neurology rotations, I was passionate about learning and I also recognized that in chiropractic school, whereas my education in New York chiropractic college was good, it did not provide me with what I really needed to know to help my patients to be an excellent diagnostician, an excellent treater of patients with the various kinds of problems they came in with. And so I realized that if I'm going to gain that knowledge, I'm going to have to do it on my own. And so I did, I spent a lot of time reading the literature and studying books and I traveled abroad to the Czech Republic on two separate occasions studying under Dr. Vladimir Yanda and Dr. Carol Levitt and I studied with them here as well in the States when they came here and studied under Dr. Janet Travelle and a wide number of people just trying to gather and gain as much knowledge and experience and skill as I possibly could. That would help me be the best as it turns out spine doctor as I could be at that time. I didn't see myself as a spine doctor per se. I saw myself as a musculoskeletal doctor but I realized as time went on and I'm sure we'll get to this that primary spine care was my real passion. Let's get into that because that is one of the big topics that I'm interested in at this present time. Explain for my listeners, what is primary spine care? Primary spine care is a new role that we're introducing into the healthcare system where in a practitioner, a primary spine practitioner plays the role of primary care for patients with spine problems. So this person needs to be capable of managing the majority of spine patients without the need for referral but also knowing how to manage the case, manage the situation, be the quarterback of the spine care team which means keenly understanding differential diagnosis, knowing when to recognize when somebody needs special tests or specialist consults, knowing and identifying when somebody needs invasive procedures such as injections or surgery or intensive procedures such as multidisciplinary pain management, recognizing when somebody requires psychological or behavioral intervention and following up with that patient to make sure that they maintain on track, they keep themselves on track toward resolution. So literally being the primary care practitioner for patients with spine-related disorders. So we mentioned before we started recording how there's kind of a spectrum between the schools, a lot of schools have different approaches and some of them really brand themselves as training primary care practitioners. But I have to say I have some reservations myself about whether the amount of experience we get really qualifies us. I'm curious what your thoughts are. Does the chiropractic curriculum as it stands provide deep enough training to allow students that are graduating to be that primary spine practitioner? To be the primary spine practitioner or the general primary care practitioner? Maybe both. I guess I consider them somewhat the same. In that you do need to have that broad knowledge of differentials and when to refer out. Maybe take one at a time. Okay, so does chiropractic training prepare students to be primary care practitioners similar to the family medicine doctor or the general internal medicine doctor? And the answer is emphatically and absolutely not even close. My primary faculty appointment is at the Medical School of Brown University. Most of my appointment is in the family medicine department. Most of my teaching is either family medicine residents or internal medicine residents. I get to interact with real primary care doctors and real primary care trainees training in real primary care. And chiropractic education is so far from what is required to be a true primary care doctor that it's not even worth having a conversation about except the fact that there are many chiropractors who think they are trained to be primary care practitioners and many schools who think they are training primary care practitioners. And that was my experience. Certainly my school emphasizes that but I remember having a discussion at one time with one of the faculty that, you know, when I was an undergrad I had a roommate who was a nursing student. This was pre-nursing and he had to do, I think it was at the time 100 sets of idols. Heart rate, blood pressure, maybe even listening to heart sounds although I don't think nursing students do that very often. But in my experience going through chiropractic school I think our numbers were 10, 20 sets of idols and I just didn't feel like I was getting the experience with it that I needed to be able to confidently tell other doctors and patients that I can be your primary contact. Right. Right. And if you speak to people in the general healthcare community even those that are open to chiropractors if you even mention chiropractors as general primary care practitioners you'll either get a blank stare or a hearty laugh. Or very concerned looks. Or very concerned looks at that very good point or very concerned looks because that is so, the notion is so far-fetched. So what about chiropractic students coming out of their three or four years of training being prepared to be the primary spine practitioner? Okay. So now we're talking. So chiropractic training prepares students to be prepared to become primary spine practitioners. Okay. Okay. So the chiropractic students gain a lot of the basic knowledge, a lot of the basic skills that are necessary to be a primary spine practitioner but are not sufficient to be a primary spine practitioner. So additional training, additional knowledge, additional experience is necessary to really become a bona fide primary spine practitioner. But of all the healthcare schools out there, chiropractic schools are the closest to providing their students with the knowledge and skills that are needed to become primary spine practitioners. But I have the opportunity to, I have a regular externship at my facility and so I've been training interns from several different chiropractic schools for the past 20 years or so. So I have a pretty good experience and these are the most highly motivated chiropractic students because they're willing to travel and find lodging to spend 10 to 15 weeks with me. The most highly motivated students and they're not, they're not, I shouldn't say they're not close. They are fairly close but they do not have the knowledge and skills to serve as a primary spine practitioner. They're closer than anybody else in terms of healthcare schools but they don't have all that it takes. What will that training look like? Is this another diplomat program of 300 hours? How will a student or current practitioner become prepared to be a primary spine care practitioner? As it stands now, I'm in the process of we're just finishing up and getting ready to launch a course through the University of Pittsburgh that is a one-year course that provides additional training to be able to serve in this role of primary spine practitioner. It consists of five modules, I'm sorry, five units we call them, five units each unit consists of a weekend seminar and a series of online courses. It takes place over a year and there's a unit exam at the end of each unit and then there's a final practical exam at the end of the course and this will be the beginning of the official certification process for primary spine practitioners. So we're doing it through the University of Pittsburgh but also it's in a loose partnership with Southern California University of Health Sciences which is where Los Angeles College of Chiropractic is. And so this is the beginning of the formal certification training of primary spine practitioners. Ultimately primary spine care training has to be, what will have to be a residency-based training but that's down the road, that's what we're looking toward but you have to start somewhere so this is where we're starting. Will this training be available to current students or is this only a postgraduate opportunity? It's designed for postgraduate so to be eligible for the certification one has to have graduated from a healthcare institution and be able to have the basic knowledge and skills required to then be augmented to become a primary spine practitioner. Will students be able to start the coursework before they're graduating? Currently the, and I'll preface this by saying that I'm responsible for the content and the delivery of the information. I'm not an official at the University of Pittsburgh so policy will be established by the university but currently the plan is for this to be a postgraduate course just as a medical graduate who wants to become an internal medicine physician or a neurologist will need to graduate medical school and then go on to additional training. It's the same thing, you need to graduate from chiropractic school in order to then go on and continue your training to be a PSP. The University of Bridgeport recently held a panel that was shared on Facebook about the primary spine practitioner initiative or goal of many to kind of start this specialty and needless to say there's a lot of controversy I guess you could say that might be a strong word but a lot of people have concerns and one of the main questions is will labeling chiropractors as primary spine practitioners limit their scope? That's the most common concern that I hear from chiropractors that it will be limiting and it's kind of funny because for one thing chiropractors currently see about 10% of the population pretty much 100% of the population have spine problems at some point if you consider back pain, neck pain and headaches so I asked the question which is limiting 10% or 90% so it's really kind of amusing to consider this limiting the other thing I would have to say is that I've been practicing as a primary spine practitioner for pretty much most of the last 20 years I've been in practice 29 years and I'm now in charge of a program an integrated spine care program at a large hospital system and where primary spine care services are the main focus of the program and I love seeing patients with spine problems spine care is my passion I try to avoid extremity problems even actively trying to avoid seeing patients with extremity problems I have plenty of them come in I have patients come in referred by ear, nose and throat doctors who have oral facial pain I have patients referred in who have shoulder pain, knee pain and I love seeing the oral facial pain patients but the extremity patients I don't have much of an interest in that and even trying to avoid those things I can't avoid them, I get plenty of them if I wanted to pursue seeing more extremity problems I would be inundated with them so in reality if a chiropractor establishes credibility in their core area of primary spine care they will then be able to leverage that credibility into other areas so not only is it not limiting it's expansive and that's what has been my experience Very interesting so you've mentioned how eventually the goal is to have this almost as a residency and I have a listener question this is from Isaac who asked what is the best path to getting more DC's hired in the mainstream healthcare delivery system That's a great question from Isaac and it's not an easy one to answer however basically for a chiropractor to become involved in mainstream healthcare it will be as a spine care doctor or it won't happen at all with the exception of sports medicine so sports medicine chiropractors several chiropractors have had success in incorporating themselves in the mainstream of the sports medicine world so that's a niche that's available to chiropractors that many have taken advantage of but aside from that if one wants to be a part of the mainstream health system it's going to be as a spine doctor or it's not going to be as anything so and what the healthcare system is looking for is people to be able to play innovative new roles that provide value in certain areas of medicine that currently high value is not currently being provided and so the healthcare system has never been more open to I hate to use the word alternative but alternative if I may use that term mechanisms of delivering quality healthcare and so probably the most expensive least effective and messy is of all of medicine is spine and so the opportunity exists but what we have to realize is that as it stands now saying I provide chiropractic care that means something to us it means something to our patients but in the wider healthcare world they don't quite understand what that means which is why I think primary spine care has a resonance it resonates with people in the healthcare system because that's an area that is where there's a recognized need you told me that you did your sport certification and I was just certified and I did the masters in sports medicine here at western states and that certainly was my original interest in chiropractic I was active as a teenager and that's part of my first introduction to chiropractic and I appreciate that you shared that sports chiropractic is one of the other ways that chiropractic can become more widely accepted do you see and this is kind of taking another listener question who asks what is the best way to start integrating this spine practitioner into a more traditional rehab or adjusting based office and I just want to add to that how can a primary spine practitioner continue say their passion for sports or any other interest that they may have in chiropractic well you know the primary spine care and interest in sports medicine are not mutually exclusive I mean for one thing there are plenty of sports injuries that involve the spine and certainly having credibility as a spine practitioner will be helpful in terms of being a recognized expert in those kinds of problems but again having a sports oriented primary spine practitioner I don't see that being mutually exclusive at all excellent that makes me very hopeful now you also have written two textbooks on spine pain on this idea of the spine practitioner what led to writing these books well as I went along my journey of recognizing the limitations of what I knew and what I was able to do in helping people overcome spine related problems I started reading the literature I started taking seminars I started studying abroad etc and I realized that there was a lot of good evidence-based information out there but it was very disparate it was all over it and there was no connection between all of the different models of care all of the different information and research that was available in the field and so I said we need to have a way to bring this all together into a cohesive model that can be applied in a busy practice environment and so as that evolved I ended up writing a textbook in the late 90s called conservative management of cervical spine syndromes and that began the process of my culminating all of this information into a cohesive approach as I developed that approach I said well you know does this approach really work in the real world and so I started doing observational studies looking at groups of patients and treated according to this approach which I had a name for it that I won't bother you with because it was kind of cumbersome name but observationally at least what I found was that yeah patients seemed to benefit from this approach and so as I started formalizing this process and started, I wasn't alone in this conceptualizing this primary spine practitioner role I and a team of others who are co-authors on my original paper started culminating this new role this primary spine practitioner this melded well with the clinical approach that I was putting together that considered all of the biological, psychological and social factors that can contribute to the spine pain experience into a diagnostic and treatment model that could be again applied in a busy practice environment so I developed this clinical reasoning process by which the spine practitioner can again consider the multiple factors that can contribute to the spine pain experience figure out which of those factors are contributing in this particular patient's case and how can I formulate a diagnosis from that and then formulate a management strategy that is most likely to respond positively to the diagnostic factors that I identified and so I call them the CRISP protocols clinical reasoning and spine pain and so I ended up writing these two books one is primarily focused on the low back and the other is primarily focused on the cervical spine with a chapter on the thoracic spine and these became basically the operations manual for the primary spine practitioner as a matter of fact the second volume of the textbook has two chapters on case studies in primary spine care where I go through 18 cases and I go through the process from the first presentation of the patient how the primary spine practitioner would look at that patient, evaluate them interact with them establish a diagnosis decide on a management strategy and then guide that patient from having this pain disability and suffering experience to resolution of the problem and all of the different things that has to happen on that process again spine pain is multifactorial there are biological factors somatic there are neurophysiological factors all occurring in the social context in which the patient lives and so the primary spine practitioner has to be able to look at all of those things figure out which of those factors are playing a role in this particular person's suffering and how can I address those and help them overcome this problem whether it's things that I can do myself whether it's things that I need to obtain from other members of the spine care team but I am the one who's responsible for guiding the patient along that process toward resolution and so the books put that process together clinical reasoning and spine pain or the CRISP protocols put that process together and help the spine practitioner utilize this clinical reasoning process in practice the first volume I've already read through and it certainly helped me clarify just the framework in my mind about how I was approaching a patient and I came out of school I think knowing all of these different pieces I certainly knew a lot about diagnosis a lot about treatment soft tissue adjustment but I didn't feel like I knew how to fit them all together in a cohesive way I think that's really what these textbooks have helped me with and I think a lot of students have had that same experience so I really appreciate it and that is exactly what it was designed for is to I have these skills I have this knowledge how do I apply what do I do in what situation when and how do I make sense of this whole situation and how do I respond utilizing the skills I have you mentioned your co-authors and some other I noticed you you had a long list of acknowledgments in both textbooks and I'm curious who you've already mentioned Yand and Levitt who are some other influences that have really informed your practice and guided you along the way well one of the biggest ones in terms of the overall view of healthcare and primary spine care and where the PSP can fit into the broader healthcare world is Scott Haldeman he may be familiar with him he he helped me to crystallize the evidence based approach to spine care but also fits in the broader scheme of the healthcare system he was extremely influential and still is on my thought process with that regard and again my learning journey over the past 30 years has been so eclectic that there are so many different people that influence that process but certainly I mentioned Dr. Joe Ferrissie who taught the neurology course I learned a tremendous amount about neurodiagnosis from him and he helped me to see where neurodiagnosis plays as an important role in spine care I mentioned in the book Yand and Levitt I mentioned earlier the scheme of people that I work with implementing the integrated spine care pathway that I'm currently implementing in the hospital system where I work that have been very very helpful to me in broadening my view of where primary spine care fits in to the wider spine care world people like Dr. Brian Justice Dr. John Ventura Dr. Kowski Dr. Tom Nooner they're my partners in this process of bringing this integrated spine care pathway to other hospital systems payers and other healthcare delivery systems where primary spine care services and integrated primary spine care pathways can be implemented and really respond to the value model that is so important in the coming healthcare system Michael Schneider Dr. Michael Schneider is my partner in the course at the PSP course at the University of Pittsburgh he's been a very important person in this process Mark Laslet is a physical therapist from New Zealand who has done so much work on diagnosis identifying pain generators in the spine and using an evidence based approach to in a credible way identify pain generators and Robin McKenzie who developed the McKenzie approach also has been very influential in that regard in terms of diagnosis and helping to bring to teach people how to take care of themselves boy there's such a large people that have been an influence and I hate to leave anybody out well I definitely will refer everyone to the acknowledgements in both textbooks if you don't mind I'd kind of like to come back to Dr. Scott Haldeman because I am a big fan he's got world spine care which I'm a volunteer with but as I look at I guess my influences or the people who I look to and Dr. Haldeman certainly is there you have made the list recently with the textbooks I'm noticing some similarities and I read through Haldeman's biography and two things I would I guess I would like to ask you about number one you seem to be involved in a wide range of things and I'm curious how do you personally manage to do so many different things and to do so productively oh wow well I think I'm very good with organization and time management so that's really the key to getting a lot done in a normal 24 hour day I'd love to have my days be 36 or 48 hours long but unfortunately they're 24 hours like the rest of us but being organized and being a good time manager is extremely important and you know I'm driven by passion and that's the biggest thing if you have passion you figure out a way the other similarity that I see and again going back to your CV with a long list of articles is that again both you and Dr. Haldeman have published a lot is writing something that you've always enjoyed doing or have done throughout your life yes ever since I was a kid I was a writer I used to write stories I used to write believe it or not this sounds awfully geeky but I used to write book reports if we were assigned a book report in elementary school I would write three instead of the one that was I just always love to write and that was just something that's in me and so that certainly has helped I talk to a lot of people that have trouble writing sitting down and writing what's on their mind and I just happen to be the type of person that writing comes very natural to me I definitely am the opposite the book reports were the hardest thing for me to get through in elementary school and I remember one day in particular we had a journaling assignment where we just had to write what did you do over the weekend and I ended up staying at least an hour after school because I just couldn't get it out do you feel like writing has led to your successes does it play a role in understanding spine care and spine pain absolutely one of the things that I find is that through writing that is the way which I bring my ideas to fruition I bring what's kind of floating around in the back of my head to the front and allows me to really communicate to myself as well as to others what's on my mind and what what I've learned and how I can put it all together so I find writing to be very very helpful in that regard and my writing has been helpful in my career because when your name is out there and your work is out there and if fortunately people find your work to be of high quality and beneficial that brings you recognition that can be brought through personal communication but having publications out there are really helpful in that regard well I do have a patient scheduled in just a little bit but I would be remiss if I didn't get through some of the other listener questions that were submitted Jerome asks on Facebook does the audible release play a role in outcomes? I'm assuming he's talking about the cavitation with spinal manipulation that's a good question so manipulation is a very useful tool it's not the only tool and I encourage chiropractors to avoid attaching their identity to that one procedure rather than attach their identity to helping patients overcome problems being a problem solver so I'll start with that the literature is pretty clear there's been a number of studies that the audible release itself is not a necessary component in order to bring about benefit of manipulation my experience is that it's highly individual that if you take if you look at a population of patients and you treat one population with a high velocity technique that brings about an audible release and another population with a low velocity technique that does not bring about an audible release pretty much both groups will do just as well both benefit equally but there are individuals within each group that may benefit from a cavitation technique whereas there are other individuals that may benefit more from a non-cavitation technique so I think it's essential for chiropractors to be good at both be good at cavitation techniques be good at low velocity non-cavitation techniques Dr. Dean Smith asks and I think this is very helpful for reason graduates with what you know now how might you approach chiropractic school and practice differently as a new graduate compared to the way you originally did oh wow that's a great question how would I approach well I would definitely be more focused I would definitely I would have my again my self identity as I just mentioned self identity placed firmly in the area of I'm my purpose is to help patients overcome spine related disorders rather than place my identity on a certain set of procedures that frees you that's a very freeing process because if you're just here to help if you see your identity and your role as just helping people then whatever you do to help people is what you do and if somebody disparages manipulation that doesn't reflect on you personally you don't have to take that personally because manipulation is simply a tool that you use to fulfill your real mission and that is helping people so I think I would that would be my the main thing I would do differently is really focus my my identity and my approach on on helping patients rather than on who I am and what I do lastly I'd like to ask for those who may be considering chiropractic school what do you feel is the most important thing students can do when choosing to go into chiropractic and then choosing a chiropractic school well I think number one being realistic and recognizing that the chiropractic profession is in the is in a maturation process as a profession we're in the process of we're in our adolescence and we're trying to find ourselves if you remember the painful period of adolescence you remember you were trying to find yourself and you were conflicted and it was a very turbulent period and perfect was that it was an awful time awful but a time of opportunity because you came out of that with a crystal clear identity of who you are and so chiropractic is currently in its adolescence and we're I think we're going to come out of our adolescence having a clear vision of who and what we are what we have to offer but right now we're in our adolescence and I think we need to be realistic about that and it really worked toward maturing and becoming an adult profession and that's not I don't mean that disparaging by the way that we're immature babies as a profession that's just the natural evolution of professions. Well Dr. Murphy thank you so much where can students learn more about you and about the primary spine practitioner process there will be a web presence with the University of Pittsburgh course to learn more about me and about primary spine care in general I would encourage them to visit the website of spine care partners which is a company that I'm a part of if you just Google spine care partners and the primary spine practitioner network there'll be information about me and about primary spine care there and I'd be remiss but if I didn't say my books have a lot of good information about both as well I highly recommend the books I'll include links in the show notes for students to find those directly on Amazon as well as links to a lot of the other things that we have discussed today as well as a collection of papers that discuss primary spine care from a number of different viewpoints well thank you so much I have so many other questions I would love to get to so maybe we can find a time in the future to continue this conversation that'd be great thanks don't forget to get your unlimited data backup for your Mac or PC that charges $5 a month by going to exploringchiropractic.com slash backblaze hey I greatly appreciate an iTunes review please go to Exploring Chiropractic on iTunes and leave a review with your comments and thoughts about the show you can also contact me at exploringchiropractic.com on Facebook, Twitter and Instagram at Exploring Chiro


So, this is module 3 where we're going to take the results of modules 1 and 2, basically our alignments and try to calculate expression and differential expression estimates from that data. So, learning objectives are to talk about how we generate these expression estimates for known genes and transcripts. We're going to talk a little bit about FPKM versus raw counts. We're going to introduce the differential expression methods and then we're going to talk very briefly about some of the downstream interpretation issues, which are really the topic for a whole another course. So, we've already seen a view like this, but this is the basic concept of RNA-Seq when it comes to expression analysis. We're starting from alignments, that's our raw data at this point. We're looking for cases like this where we have, based on the coverage track, different amounts of expression between different samples, and we're also looking qualitatively at what transcripts are we seeing evidence for expression of. This is a case where we're seeing some down-regulation, and I just pointed out the 3' bias that Malachi described earlier is quite apparent here, so the coverage really tails off as you get away from the 3' end. So, FPKM and or RPKM you're going to hear a lot about. This is the most common measurement that's described for expression analysis of RNA-Seq. It was kind of invented right from the first days of RNA-Seq, or really even earlier back in the days of SAGE we had a similar concept. They're basically the same thing, it's really just a terminology, reads or fragments, per kilobase of transcript per million mapped reads. Of course, there are a lot of different ways to arrive at these values. Conceptually they're the same, but in the details they can differ quite a lot. They're based on the concept that in RNA-Seq the relative expression of a transcript is proportional to the number of cDNA fragments that originate from it. But we have to be aware of this problem that the number of fragments is biased towards larger genes. So if you're just randomly sampling, you take a larger gene and break it into pieces, you're going to get more reads from that gene just because it has a bigger space. And we don't necessarily want that to be reflected as a higher expression level of that gene. Also, the total number of fragments is related to the library depth. So the more you sequence, the more your fragment counts go up, and apparently your expression levels go up, but that's not really reflecting any change in the underlying expression level of that gene in that sample. It's just being sequenced more deeply. So these two measures, FB-CAM and RP-CAM, are an attempt to simultaneously kind of normalize or scale for gene size and library depth so that you have some kind of apples-to-apples comparison between genes and between samples. Yeah? No, sorry, it's RP-CAM, slash FB-CAM, and these are the same things, basically. It's being done in this formula. So you're taking the total number of mappable reads and you're dividing by the number of mappable reads in the library, the total mappable reads in the library, and the number of base pairs in the gene transcript X on it, et cetera. And this 10 to 9 is actually taking into account a thousand bases and for... it's giving you the per thousand bases and per million fragments. So the gene size is factored in through this L value. So idea being that either... so RP-CAM or FB-CAM, it's just reads versus fragments. This was... basically came about when we started to do paired-end sequencing, we didn't want to call it reads anymore because we're counting two reads as representing one fragment. And you'll see this much more commonly now, RP-CAM, you don't hear discussed as much. And it's simultaneously trying to account for both the gene length and the total depth of the library. And there's more than just these two, but there's a bunch of biostart posts where they go into great discussion about what an FB-CAM really is and what does it mean. So cufflinks is a really complicated method. This paper, or figure from their paper tries to summarize it, simplistically it's much more complicated than this even, and even I find this figure fairly complicated. But the general idea is you're taking alignments, you have these bundles of alignments which are basically assemblies of reads. And those assemblies of reads are mapped into a graph, an overlap graph, and then transcript isoforms are inferred by the minimum number of paths required to cover that graph. So this is not a perfect method, but based on having a bunch of reads in an area, making an assembly of them and then saying, what's the minimum number of transcripts paths that could explain this graph? And based on that they define these transcripts and they assign abundances to each of those isoforms using a maximum likelihood probabilistic model which is also very complex. So if you want to know the nitty-gritty details you really have to read the cufflinks paper and probably take a statistical degree. But in the end they produce basically their own concept of the transcriptome and we're going to run cufflinks in a few different modes and so you can basically try and guide it to use our current known idea of the transcriptome from something like Ensemble or you can run it in a totally de novo mode where they just infer all the transcripts in your samples without any preconceived notion of what the transcriptome should look like. And there's sort of some modes that are in between those two extremes. And it tries to be clever and make use of information like the fragment length when it's assigning each read to each one of these putative transcripts. So it'll say okay if this read spans, looks like it's spanning this many introns going from junction X on 1 to 5, is that likely given the fragment size of the reported fragment size of this library. And it uses other pieces of information to assign reads to the most likely transcript and then come up with estimates per transcript. So it's much more sophisticated than say a lot of the well-counting methods and it generally works pretty well but it's not perfect either. From that you get these cufflinks-based estimates in the form of FPKMs for all the transcripts in your transcriptome which has been inferred by some process of assembly. And then cuffdiff basically takes those values and it models the variability in fragment counts for each gene across the replicates if you hopefully have replicates. And then the fragment count for each is estimated in each replicate as before along with the measure of uncertainty that arises from ambiguously mapped reads. So transcripts with more shared exons and few uniquely assigned fragments have a greater uncertainty and that makes sense. So basically if there's two transcripts and they're largely the same and this happens all the time, this is why it's such a challenging problem and maybe they just have a very slight difference in the last exon. There's a lot of uncertainty. So a read that's mapping to exon 1 which is used exactly the same way in these two transcripts. Have no way of knowing whether that read should have come from transcript A or transcript B or should like isoform A or isoform B. So it tries to do its best with those areas where there is unique content or different junctions that distinguish the transcripts. But at the end of the day the more similar set of transcripts there are the more uncertainty there is in which transcripts isoform or sorry the fragment comes from. And that is incorporated into the model or into the algorithm. So it combines these estimates of uncertainty as well as the cross-replicant variability that it observes into what they call a beta-negative binomial model of fragment count variability to estimate count variances for each transcript. So now you've got estimates of FPCAMs of intensity or expression levels and then some concept of variance between samples for those transcripts. And those variance estimates are used for statistical testing to identify differentially expressed genes in transcripts. And again this paper tries to outline this method and or this figure outlines a method and the paper goes into much greater detail. So if you want to really understand how CUPDIF works become a statistician and then read the paper. Cuff merge is a step we're going to run and it's necessary because of this thing I mentioned where cuff links tries to infer the transcriptome. So it's going to take your two samples in the simplest case where we're just comparing sample A and sample B and it's going to say looking at this data for the most part I'm not going to just believe what ensemble says are transcripts I'm going to infer what the transcripts are and then it's going to do the same thing for sample B. But the problem with that is of course it comes up with slightly different answers even if they're like totally technical replicates it won't come up with exactly the same structure of the transcriptome. So then when you're trying to do a comparison between the two you're not really comparing apples and apples. So cuff merge is what makes an apples to apples comparison possible. Basically merges together those two concepts of the transcriptome structure to create one consistent concept so that a direct comparison can be made. And you can optionally provide a reference GTF which we're going to do so that when it's merging these isoforms that it's inferred it also takes into account known isoforms. So that where possible you can assign expression and differential expression to genes that we already know about not just potentially novel transcripts. Yeah. So it'll be, you're talking about at the cuff merge stage, it'll be okay, it'll take the transcript as evidence of a transcript from the one sample and it will have that concept of the transcript in the merged GTF file but it will be assigned a zero value from the sample which doesn't have any evidence for that transcript. So Cumberbund is our package that the tuxedo suite has developed for visualizing and summarizing the results of their differential expression pipeline. So one of the things you're going to see when we run cuff diff and cufflinks for that matter is that it produces this huge array of confusing files. There's files at the level of isoforms and CDS and gene and other levels and each of those has multiple different levels, FBKM files and retracking files and so on and none of them are really actually convenient for proceeding with your analysis. So usually what happens before Cumberbund is you'd go into those files and you'd have to kind of munch them and reformat them and combine them across different folders so that you would get something that looked kind of like your gene expression matrix that you would get in an old microwave experiment. And we still do that often but an alternative to that is to take Cumberbund and basically just point it at the differential expression folder and it underneath has the knowledge to assemble the data in a useful way and it can create all kinds of useful things like distribution plots and correlation plots, classic plots like MA plots and volcano plots. It will do clustering in PCA and MDS analysis for you. It can make heat maps at a global level or for as few, all the way down to one gene or just a selected subset of genes or just a significantly differentially expressed genes and so on. And you can also create some pretty nice gene and transcript level plots showing the transcript structure which is useful especially where cufflinks is defining its own transcript structure and the expression level of those transcripts and genes. And it's things like this. So there's like a scatter plot of normal versus tumor. There's distributions to see what's the overall distribution of the data look like. How do the samples cluster compared to each other? Typical heat map of genes versus samples for the most significantly differentially expressed genes. Expression levels for a particular gene broken down by isoform. So this gene we looked at before, TST, it predicted four isoforms for that gene and most of the action is happening for this one particular isoform which is being down regulated from normal to tumor or vice versa, I can't remember. And things like this where you actually get to see the transcript structure of those four transcripts. You can compare that back to what ensemble has in terms of transcripts for that gene. Look at other tracks that you can load in for UCSC like conservation and plot it against an ideogram. So it can do a pretty good job of making sort of publication ready figures. So we're going to go through the whole tuxedo suite and I recommend it. It's relatively easy to run and it's very powerful especially if you need to get something together quickly. But it's not everything. A lot of people don't believe that FBKM estimates are perfect. They don't believe the transcript predictions are perfect and some people don't believe the statistical methods that are being used by the cufflinks package are good. So a lot of people are going back especially just for the differential expression analysis to these raw read count-based methods which they think are modeling the data better. So instead of calculating an FBKM, you basically just assign reads or fragments to a defined set of genes or transcripts and we can define those using a GTF file and then we can just determine raw counts. So basically just how many reads we're seeing mapping to gene X or transcript Y. Doesn't that sound like some of the same bias that FBKM trades to normalize the gene length? Yeah so the reason that that is okay is because people typically only use this for a differential expression so where you're comparing two samples where they're going to have the same gene sizes and they're going to model the library depth as part of the statistic. So don't worry about the gene size because you're always comparing two things with the same gene size in the statistics and they do account for the library depth issue. So those things are taken into account and they claim they're being modeled better using these negative binomial distributions or cross-on distribution statistics than they are with things like FBKM with cufflinks and they're pretty popular. So the problem is of course you need to get these raw counts. We're going to show you how to do this with one piece of software called HTC Count but there are others and that's just an example of what it looks like to run but we're going to go through that. There's an important caveat to this and there's a discussion of that on seek answers and this is basically that the counts are only as good as the features that you define to count for and it suffers from exactly the same problem as I was describing for cufflinks which is that if two transcripts are exactly the same how do you know which transcripts, so like let's say a gene has two transcripts and they're exactly the same except for one tiny little axon at the end. Most of the reads are from over here, maybe all of the reads that mapped are from these regions of ambiguity between the two isopharms, you have no way of knowing where to count that to any particular fragment. And things like HTC Count tend to make simplifying assumptions, they usually bin those as ambiguous and don't count them. So at the transcript level it's going to work okay for some transcripts and really bad for some others and I think in this form you even have the author of the tool saying this was designed for gene level analysis, I don't make any claims that it's super effective for transcript level analysis. So they're kind of side stepping in some ways the problem that cufflinks is trying to deal with but it's a difficult problem and they may not be dealing with it perfectly or very well at all. So yeah doing things at the transcript level especially where transcripts aren't very distinct from each other is still very challenging. So one thing that's confusing with HTC is that you still have to normalize, you still get and you can never list the number because the number you're laying is like 200 million or something so you always get like a transcript per million pieces mapped or so it seems like it's very similar or because of my, because I have very low experience in this it's very different. So it looks like the two methods are... How are you talking about using the data I guess that's what I would ask. So HTC is a read count method, right, basically for the gene but in it you still have to normalize to the number of sequences you read in the lane and so you still have to go through a normalization procedure. It depends what you're going to do with the data so if you're using something like HR or one of those differential expression count-based packages that would be like that's part of the thing they handle. But if you want to like take those read counts and do something with them like display them and heat map and compare two genes then yeah you're back to the same problem of how like having to normalize for gene size or having to normalize for library size. I guess the only advantage is that it has an excellent level of cameras. For me the only advantage it has is that you can use it with the count-based statistics. That's the only thing I use it for. If I want to just say something like does this gene have a higher expression level than another or if I want to make a heat map I'm right away going back to some kind of FBKM. And then yeah why not maybe just use cloud size I don't know. Unless you yeah unless you it's basically a more simplistic you can still you know normalize for relength and you can basically calculate your own FBKM from the HTC counts using your own definitions of genes or transcripts. Is it possible to do a new set on FBKM on an excellent basis? Yes it is. But it has the same problems as described for transcripts. I mean well because exxons also can overlap right. Maybe less than the transcripts but you definitely can. So what are the advantages like basically what should I do? This is what we do. We typically use FBKM when we want to leverage the benefits of the tuxedo suite when we want to do things like visualization for calculating fold changers etc. And we only use the counts for when we want to make use of these more robust statistical methods for differential expression. Especially because they seem like they're better at accommodating more sophisticated experimental designs. So like the EDJAR and DC methods you can set up like really complicated experimental designs where it's not just like five tumors versus five normals. And it's not clear that cuff diff is quite as good at that. But all of the places I've seen where people are using DC or EDJAR with count based they still tend to be going back to FBKMs for something. They're using it for filtering it or for calculating fold changes or for display purposes. And I guess the question is do you want to get FBKM on your own or let cufflinks give it to you? We usually let cufflinks give it to us. So I mentioned we can get these raw counts and I've just listed a couple of the packages. We're going to do a really quick tutorial of how to basically use EDJAR for these raw count approaches. But there's another one and there are lots of others I think for several others if you search for them. It's probably still advisable to not trust just one for differential expression. This is actually from the cuff diff to paper. They showed that most of what they predicted as differential expressed in their test dataset from cuff diff was detected by one or more of the other methods which they looked at EDJAR and DC. But there's definitely some substantial amount that are being predicted by one and not the other. And they didn't say which of these they think are true and maybe they're all true. So if you don't run EDJAR, you're losing all 87 of these potentially interesting observations. Or it's more loose. We don't know if these are good or bad. But yeah, it seemed to be at least the most comprehensive in terms of the total number passing. Also I would guess it depends on the P value that they heard there on our sign and also the fold change. Exactly. I would presume for this they've tried to set like a comparable P value and fold change but they're using different underlying statistical methods. So it could be that like a lot of these were just barely significant in DC and just barely not significant in cuff diff. You'd probably want to do like a more comprehensive analysis of how they differ. But if you want to just, it depends if you're in a hypothesis generating exercise you want to make sure you're not missing anything. It might not be such a bad idea to try more than one method. And maybe you have more confidence in these guys that are predicted by multiple methods. So one thing that we have to remember and so far the community is generally not doing a good job of in RNA-Seq is that the lessons learned from microarray days are some of them never learned but should have been learned. And this paper gives an amusing review of this issue which is basically sequencing technology does not eliminate biological variability. So you still have biological variability and you still in order to find significant events you need replicates. Probably almost nobody is doing this but it's recommended and the Marth Lab has actually developed a tool to help you do power analysis for your RNA-Seq experiments. So they take as input I think things like total library depth and read size and so on and the estimates variability and tell you approximately how many samples they think you need to run for a particular kind of experiment or what depth you need. Would that be mentioned a little bit in the intro? It might be. I mean for sure it talks about the issues of needing biological replicates but I don't know if it talks about power analysis specifically. So we do need biological replicates for RNA-Seq and the study design matters. So there is a bit of a tendency to just run RNA-Seq experiments without always thinking about what you're doing or why you're doing it. And there's some good BioStar posts on these topics. Another lesson learned which is more important than ever is the issue for multiple testing correction. So as you compare more features it basically becomes more likely that the treatment and control groups will appear to be different for at least some number of features by random chance alone. This is basically the simplistic explanation of the multiple testing correction problem. And we've known about this for a long time so it's well known from array studies where you have tens of thousands of genes or in the later days hundreds of thousands of exons that you need to do multiple testing correction before you can put a lot of confidence in a significant differential expression result. But with RNA-Seq it's more of a problem than ever because now we have essentially all the complexity of the transcriptome. Not just what we thought to put on the array and there's almost an infinite number of potential features. So you can look at things at the gene and transcript level, you can go specifically to the exon level, you can look at junctions, you can look at retained introns, microRNAs, link RNAs, et cetera, et cetera. Basically like Malachi said the whole genome is essentially transcribed at some level and we can look at all those levels and we can conduct tests at all those levels. So there's just a massive, massive number of tests. So you really need to think about issues like test reduction and multiple test correction when you're doing your analysis. And I usually use the bioconductor malt test package. I find it's pretty good for doing both like the really simple single step corrections like Bonferroni or Benjamin Hopper or if you want to do something more sophisticated there's a function called MTP which has quite a few statistical tests and it does like a random permutation approach which is pretty good. The multiple testing correction, the COFTIF does its own multiple testing correction. Yeah, exactly. Would one prefer this? I would be probably at least preliminarily satisfied with what the COFTIF is doing. They seem to know their business. I was thinking more when you get into doing analysis outside of the COFTIF framework. I'm not sure. I think EDGE-R might also have built-in multiple testing correction. So they're obviously aware of these issues. Downstream interpretation of expression analysis, we're just getting through the point where we have the data to start this is already this whole workshop as you can see. So it really is a topic for another course and there are I think CBW courses that cover a lot of these topics. But what you get out of this pipeline is expression estimates and differential expression lists and those can be fed into many or most of the analysis pipelines that you would use and have used for many years from your microarray experiments. We do provide a supplemental R tutorial on sort of how to format cufflinks data and start manipulating it in R. We're not going to go through that today, but you can take that home if you want to start developing your skills beyond the kind of canned analysis that's provided with Cumberbund. Cumberbund does do some downstream analysis, so it lets you do things like create heat maps. But if you find that the heat maps that it generates are not to your liking, there are a number of other R packages which you can use to do your own custom analysis. For things like classification analysis, a lot of times we don't really have the sample sizes yet or the clinical details, but this is changing. So for projects like TCGA you're starting to see hundreds or thousands of samples for a particular tumor type and sometimes you're getting clinical annotations and we can start designing prognostic signatures from RNA-seq data in the same way we have been from microarray data. So if you're interested in that, I suggest WECA as a good learning tool and then there are many, many R packages for classification analysis. I usually use random for us and there's a BioStar tutorial being developed for that, so you can watch for that. Pathway analysis. This is something people always ask for and never really know what to do with when you give it to them. But people will ask you as soon as you can do the stuff in this course. Oh, you've got expression data and differential expression analysis. Now tell me what pathways are significant. So David is a really easy web tool. You can basically just paste a list of gene IDs in there. It's not bad. I've used Ingenuity Pathway Analysis quite a bit. It's commercial software though. Site escape is not bad. It's good for visualizing and it does have some pathway plugins.  So if you search this link, I think it finds like 250 packages. So you probably have to like go on BioStar and see what people actually recommend or use or Google around a bit. So sorry to blast through that but we do want to get through the tutorial material. Just to remind you where we're at. We are done with getting our data. We have made our alignments and now we're going to get expression estimates with cuff links. And then we are going to use cuff merge and cuff diff to do differential expression analysis.