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hello everyone and welcome to pack bio day i know you all are probably just as disappointed as i am that we are not together down in san diego for pag um but i'm really excited to be able to host this half day virtual event for us so that we can share and discuss all things plant and animal genomes um so we've got a really excellent lineup today we've got 10 different on-demand presentations on everything from face mites to cannabis to dingoes and all of those presentations will be available on demand all day long so i encourage you to go check those out in addition we've got two live workshops um hosted at 11 30 a.m uh concurrently one focused on genome assembly and the other one focused on iso seek so definitely drop in there to hear more about those particular workflows and to get your questions answered by the experts lastly we have a general drop-in session that'll be open um so that you can get any pac-bio-general questions answered by pac-bioscientists and then lastly i'm actually hosting a live panel discussion at 12 15 pacific um with several of our speakers on the topic of the future of sequencing in plant and animal sciences i imagine it's going to be a great discussion about how we tackle strategies with genome assembly and annotation all the way into what we see kind of coming out in the future so i encourage you to join us for that as well if you have interest um and we have a really great keynote speaker for you but before we launch into that um i just want to reminisce a little bit about what we've done over the past year so it was this time last year at pag2020 that i shared with you the hi-fi sequencing data type and explained what all you can do with it and really with the goal of convincing you that you should hi-fi all the things and throughout 2020 you did not disappoint a quick scroll through twitter shows just how much data you generated and all the really great science you did and it's been really really exciting to see how well hifi is working in all of your hands um and for those of you who are newer to hi-fi sequencing here's just a quick explainer so hi-fi reads are generated by sequencing around a double-stranded dna molecule that has two hairpin adapters on on each end creating a circular molecule and as we're able to sequence around this circular molecule multiple times we can stack up those individual reeds and call consensus at each base and that uh consensus called sequence is what we call a hi-fi read and these hi-fi reads can be up to 25 kb or even longer and they actually have a median accuracy of q30 or 99.9 accuracy and so with that read length and that type of accuracy you could really imagine a bunch of different uh value that you can get from from this data type across a wide variety of applications so everything from genome assembly to high accuracy looking at variants both at the single nucleotide level and at structural variation level targeting really difficult to sequence regions or getting full length rna transcripts in order to annotate your genome or even parsing apart complex microbial communities in metagenomic samples and with the continued increase in throughput that we're seeing on our um sequencing platforms most of these applications can be done in one to two smart cells um so there it's really a cost-effective way to get it biological questions and if we look at uh each of these applications um what i mean by a high quality genome assembly is that you're actually able to get extremely high contiguity you are getting more of the genome represented within the assembly because you're able to get through repeats you're getting very high base accuracy and making sure that all of the genes that you're getting are in frame the hifi data type actually has significantly smaller file sizes than um noisy long reads and that actually ends up reducing the analysis time so you're reducing your overall project and project time and cost and then hifi data is actually pretty easy to work with because of the smaller file sizes and shorter analysis time but also on the wet lab side you're not trying to get extremely large dna fragments because you're working in kind of this medium 20 kb range which means handling of your samples is actually easier as well and because we have easy end-to-end solutions you're able to do the workflow pretty seamlessly on the rna sequencing side of things the value of doing isoseq which is pacbio's long read rna sequencing is that you're able to get the entire transcript from the five prime end all the way to the poly a tail in a single reed so that you are not having to infer what the alternative splicing looks like across different mrna transcripts and you're actually able to just see the entire thing in a single reed without assembly when it comes to metagenomics you can take a metagenomic sample from anything you're interested in studying whether it be soil or water or some sort of tissue and when you do high-fi reads you're getting a lot of functional information because every reed has anywhere from seven to nine fully intact complete genes in it so that you're able to resolve things down at the strain level you're able to have more complete genome assemblies for things that can't be cultured and you have very high contact purity so it makes meta-genomic samples really easy to work with as well and on the targeted sequencing side of things um the high resolution and the long read length actually allow you to identify variants that you wouldn't be able to pick up with short reads or to have a more zoomed in approach at local genome assembly and so with all of that really great information on the various applications that you can do with hi-fi sequencing it was really cool to see some some new things come out in the last year so we demonstrated that you can assemble all things big and small you might have seen that we actually tackled the california redwood genome which is a nine gig hexaploid and with only 20 fold coverage we were able to get a very contiguous complete genome assembly and spoiler alert later today liz sang will be presenting on the isoseq data that then came out of it and the ability to use isophase in order to be able to fade the haplotypes we also saw on the other end of the spectrum that we released the amplification-based ultra-low dna input workflow which really opens up the ability to make reference quality genome assemblies for really tiny arthropods so that was really exciting and there are several talks today focused on that work as well we also saw an emergence of pan genome projects come out and this is because not only has the cost and the time for generating genomes gone down but the resolution with which we're actually able to look at the genomes because of the high accuracy means you're able to see the variation between individuals of an individual species so there's a lot of work being done there that's really exciting to track and then lastly um with the release of the sql 2e system where we're seeing that you know you can have better data with less time and more cost savings due to being able to get hi-fi reads directly off the instrument which significantly reduces your um uh your analysis time and your storage for data and so really what that means is we have full end to end single technology solutions that help us tackle some of the big questions out there in biology whether it's trying to breed drought resistant crops in order to better secure our food source for the future or protecting coral reefs from climate change and bleaching to tackling um diseases in our furry friends that might be used for translational research later or protecting us from things like murder hornets which are threatening to take out our honeybee population or giving a little helping hand to some of the world's endangered species there's really a way that hi-fi sequencing can help in all of these efforts and so with that little background on hi-fi sequencing and how pac-bio is helping conservation i am very pleased to introduce dr harris lewin he is the distinguished professor of evolution and ecology at uc davis and he's also the chair of the earth biogenome project um and this is a a large genome project that is aiming to sequence all species of eukaryotes on earth and is an umbrella under which many of the genome initiatives that you see out in the world like the darwin tree of life or the vgp i5k are a part of so he has a lot of really great experience in sequencing and understanding biodiversity and so with that i will turn it over to harris thanks so much michelle for the introduction and greetings to all of you and thank you to pack bio for the invitation to speak on pac bio pack bio day today i'm going to tell you about the earth bio genome project a little background on the project and about the activities of the earth biogenome project and its affiliated projects in the area of conservation genomics okay so what is the earth biogenome project it's a project of confederated international network of networks that has a common goal of sequencing and annotating the genomes of all one and a half million known species of eukaryotes in 10 years just a moderately ambitious goal now the project as it stands this network of networks now includes 38 member institution network nodes on every continent except antarctica and its members are committed to the development of standards for the project dissemination of best practices and coordination of sequencing analysis annotation and training activities is also a very strong program to develop ethical standards for participation in the project data sharing and access and benefit sharing the international scientific committee of the earth bio genome project is chaired by stephen richards at uc davis and the international science committee has five subcommittees chaired by internationally renowned scientists each with specific responsibilities to core areas that will benefit from standardization across all the networks and those are marijuana check who's the chair for the sample collection and processing subcommittee richard durbin chairs the sequencing and assembly subcommittee paul fletchek chairs the annotation subcommittee justin lin plateau chairs the data analysis subcommittee and xiaofeng lee from bgi chairs it and informatics and all of the standards documents will be available on the ebp website some of them are already available but all of them will be available within the next few weeks now as i mentioned there are 38 affiliated community regional and national projects in the uh that are aligned to the ebp these are all aligned to the basic uh mission of the ebp and collectively these projects cover all eukaryotic taxa and represent most of the large scale global efforts in biodiversity sequencing and then what is the status of of biodiversity sequencing well of the 12 to 15 million eukaryotic species on earth including about 8.1 million species of plants and animals there are only about one and a half million eukaryotic species that have been described and documented and have been named and less than 0.4 percent for a total of 5 700 species have been sequenced at least at some level of whole genome sequencing if you look at the level of uh the genus about 2 percent of all 150 000 genera have been uh two percent of sp uh all have been sequenced and about fifteen percent of the eukaryotic taxonomic families now the reason you have that p1 p2 and p3 there is because those are the phases of the earth biogenome project so phase one is the sequence of representative species from each eukaryotic family phase two is for each representative species from each genus and in the out years years seven through 10 of the project to sequence all species and so with at a level of point less than 0.4 our current understanding of the evolution of life on earth is extremely limited now what about the quality of those 5700 assemblies this figure which was beautifully uh made by steve richards at uc davis shows a plot of the scaffold n50 against the contig n50 for all the 5700 species in the ncbi with genome information in the ncdbi database the genomes created with short reads mostly fall within the contig uh and 50 of less than 100 000 base pairs and scaffold and 50 with less than 10 million base pairs these are the let's see if i get my yes these are these uh orange dots uh shown here while those with uh long reeds up in the right upper right uh quandary and high c scaffolding comprise the genomes with the highest continuity uh only the 410 genomes falling in the box upper the orange box to the right that's the shaded box here represent genomes that satisfy criteria for reference quality genomes while those in the yellow satisfy the criteria for for the draft quality genomes about 1600 genomes in all in the yellow box now important point is that assembly continuity is improving rapidly and this is due really to the tremendous advancements uh made uh by pac bio in their technology uh first with uh clr and more recently in the past couple of years uh and now brought to really to production level with ccs reads or hi-fi okay and this is just a little different way of looking at it this is uh looking at these uh these 5700 assemblies uh across a ten-year time space uh frame and the left panel is all assemblies and as you can see uh we're actually approaching an exponential growth phase in in the numbers of whole genome sequences for eukaryotes and this is only eukaryotes this does not include microbial genomes or bacterial genomes but as you can see the number of draft and high quality reference genomes is quite small in fact the number of reference genomes here on the right is only seven percent this is this 400 or so of the five thousand seven hundred total only uh seven percent of the total are currently of of reference quality and uh this is uh important because as we move into phase one which is the sequence of representative species from each of the nine thousand approximately 9 300 taxonomic families we're going to be producing globally about 10 reference quality genomes a day per day every day for three years to meet that goal now the capacity to do that where is is really there today and it's just a matter of coordinating it organizing it in a way so that the right genomes are done in the right place and at the right time and the exciting thing in looking at that figure on on on the right is in green is you can see this doubling of reference quality genomes in the last year to 400 now most of those genomes have been produced by ebp-affiliated projects about half of those are actually fungal genomes that have been produced by uh by jgi and that's for the thousand fungal genomes project and many of the others uh are uh coming from uh from from sanger uh in the uk and from the vertebrate genomes project at rockefeller institute uh rockefeller university under the direction of eric jarvis so these numbers are increasing rapidly and are going to really be exploding over the next three years as we go toward the goal of achieving 9300 reference genomes in three-year period all of this has been laid out in the article published in 2018 the perspective piece and one of the stated goals in the perspective piece and this is sort of my introduction to the conservation part of the talk is the protection conservation and protection of biodiversity and in that paper we specifically stated as a quote from the paper that we will sequence the genomes characterize the genomic diversity of the more than twenty thousand three thousand species at that time that currently listed as endangered by the international union for conservation uh of nature and that this was going to be a high priority and a goal of the ebp well things have changed in the last three years the number of plant and animal species that have been surveyed by ucn has increased to about 129 000 species and what was then about 23 000 is now increased to 3 5 000 more than 35 000 actually 35 775 species are threatened with extinction and that's 28 of all species of plants and animals that are surveyed 543 it's been documented 543 species have been lost alone over the last 100 years and that number 4300 if you look at the fossil record that number would normally take 10 000 years to occur accrue so these extinction rates are well above the background rates are extinction and believed to be due to anthropogenic factors uh such as climate change and habitat destruction and if you believe the models that are currently used to predict uh species extinction rates uh uh the modelers uh scientists have now uh predicted that by the end of the century we may lose up to half of all plant and animal ute species due to due to changes in the environment and to the loss of habitat now what about those species on the iucn red list uh carolyn hogg at the university of sydney has um has made this very nice uh chart which shows that there uh of the 13 hundred five species on the iucn red list that are completely dependent on conservation on management for their survival in the wild of those 13 000 which represents 38 of the species on the list right they were 35 500. um 38 of those uh require today uh conservation uh for their survival and of law of those less than one percent point eight percent or about a hundred have a genome sequence so what is the value why do we want to sequence genome why do we need whole genomes uh in the first place many conservation biologists are happy sometimes with a bunch of snips or using rad seek and this this this argument has been uh raging in in the literature for a number of years but there have been a few very important papers in the last few years one by beth shapiro and megan supple and the other by kathy and carolyn and uh paris and emma in australia that have really outlined why this is important and that you know the the important reasons are of course with whole genomes you can improve species definitions and proving the definitions of species important because it's species that you want to conserve you can improve the distinction of where the boundaries of certain uh lie that's very important for management strategies and maximizing genetic diversity between populations you reduce uh the lack and reduce the level of input inbreeding and importantly you can increase adaptability and resilience to climate change through the identification of alleles that improve resistance to drought salinity and to water tolerance now this relationship between genomic diversity and threatened species was really elegantly um illustrated by the ebp-affiliated uh project zoenoli the zomonomia consortium of which i am a part i was published just uh last month in nature and it has this beautiful figure that shows a plot of the overall heterozygosity of the overall heterozygothy of um seven endangered species plotted against uh segments of homozygosity and as you can see the seven endangered species of the 131 new species that were sequenced as part of this project the seven endangered species had they did have significantly higher stretches on segments of homozygosity and overall uh lower levels of homozygosity and all nearly all of those species appear in that left quadrant of having reduced heterozygosity and longer stretches of homozygosity and it's well known that species with lower genetic diversity have a higher risk of inbreeding depression and lesser ability to adapt to a changing environment so therefore uh genomic diversity is a very useful marker for understanding species at risk for extinction now zoonomia is just one of uh 16 affiliated ebp-affiliated community regional and national projects with a strong conservation focus i'm not going to be discussing all of these projects today i'm going to be talking about two of them and and i'm going to be giving you some details about the goals and objectives and progress of two of these uh important projects now all of the projects uh in with conservation not just the two that i'm going to speak about share one important goal and that is as carolyn likes to tell us it's bridging the gap between geneticists and genome biologists and ecologists and practitioners of conservation the ecologists and also policy makers as you can see from the slide the gap here is very wide it's still very wide and there's a lot of research training and education on how genome information can be used to promote conservation efforts because people on the right side don't quite clearly see the need for whole genomes just yet but as i mentioned this is starting to come as we begin to demonstrate the utility of having whole genome sequences for the development of policies and the implementation of these policies for conservation and so bridging this gap between the genomicists and the conservation practitioners ecologists and policy makers is critical to lowering the extinction rates that i discussed previously the first project is is i'm going to discuss uh is being conducted in australia which happens to have the worst record in the world sorry to my australian colleagues but they know this as a country for the extinction of mammals in the last century carolyn hogg kathy belov and their colleagues at the university of sydney and across australia have set their sales to do something about it based on their successes with the tassie devil now the bio platforms australia in collaboration with the university of sydney and others have launched a new program to bridge the gap between genomics initiatives and conservation management actions this program aims to provide new genomic resources for australia's and threatened species reference genomes associated population data and an online portal that can bridge that gap between the genomic knowledge and those in the field who are implementing conservation strategies the project that is just being completed by the threatened species initiative is a pilot project there's going to be much more uh in the future but the um [Music] the pilot project involves a sequencing of seven species four of them are mammals two of birds and one plant species all these species have been sequenced using back bio high five for generating long reads and all of them will meet the ebp evp minimum contact size of 1 million base pair standards so this is being finished up and the data for this project are being will be made available shortly second project i'm going to discuss briefly is the california conservation genomics project which is arguably the leading project in the world today leading conservation genomics project in the world today the project is led by brad schaefer at the ucla la crete center for conservation science and the ccgp is funded by grants from the state of california to the university of california with additional partners in the u.s federal government and private industry the california conservation genomics project will study 225 species that are distributed across a thousand different sites in california 130 distinct projects involving 71 principal investigators who are providing samples and knowledge about these species where they're located in their biology these 71 pis are distributed across 10 uc campuses uh as part of the project 150 of the 225 will have reference genomes produced 150 represent approximately all the genera in the 225 species all of them will be sequenced with hi-fi and scaffolding will be done with omni c and this will all be done at the uc davis genome center for the hi-fi sequencing at uc santa cruz and dovetail for scaffolding and assembly and uh right now there are about 70 species whose reference genome sequencing and assembly in progress the goal is to complete all 150 by the end of july of this year now in addition to the reference genomes what scientists the technical committee in the steering committee is also very important for the ultimate utilization of the data for this project is annotation and to give equal time to all competitors we must really thank illumina for their program the illumina ebp100 program which has dedicated resources flow cells reagents library kits for the annotation of 50 genomes and this will be done by providing uh you know kits for for uh library production as well as the flow cells for for sequencing both of the rna-seq libraries and of the of the uh omni-c libraries in addition to all of that for the population genetic data 150 species will have resequence genomes i'm sorry 150 individuals of the of the 225 species will be re-sequenced for population genetic data to obtain allele frequency data that will be useful in understanding the population genetics of these species and then ultimately use for management strategies the figure on the left shows the state of california with the the distribution of the thousand sites that are being sampled by the principal investigators and their uh students and colleagues now the goals of the ccgb then are to have a broad and complete coverage of uh taxonomy of species uh in the state of these an understanding of these 225 species in the state as well as their as well as ecological coverage and replicate this across for as many species uh as as possible and all of those sites all of those species and sites are shown uh here in this part of the figure quite dense sampling as you can see for marine organisms off the coast from the from the border of oregon all the way down to the border with mexico plant species vertebrates and invertebrates as well and and again as i mentioned before similar goals to develop maps of this variation across the state hot spots in the state that are resilient uh places where you have resiliencies due to uh climate change fires uh as well as uh increase in temperature salinity and so on uh to develop new policies that could connect the corridors uh to these hot spots to maintain genetic diversity and recommendations to policy makers for the acquisition of critical lands to protect and conserve threatened endangered species within the state so much of the groundwork for the california conservation genomics project has been laid at the uc davis genome center with generous support from pac bio an excellent collaboration with jonas korlak who's been instrumental in supporting the earth by a genome project and many affiliated projects around the world i'm going to show you a few hi-fi assemblies recently completed at uc davis genome center that demonstrate the incredible power of the pack bio hi-fi technology for generating highly continuous reference quality genomes and all of this is done at a cost that we could barely even conceive of just three or four years ago and i do want to congratulate pot bio on the tremendous advances in bringing high accuracy long technology to the genomics community it has certainly been a great a real game changer so the first species i'm going to talk about is the western pygmy blue butterfly this is north america's smallest butterfly and arguably one of the smallest butterflies if not the smallest butterfly uh in the world it is not a an endangered species but it does occupy some unique ecological uh niches in the state that make it an extremely interesting species to study so this is a collaboration with adriana briscoe and you see irvine and her colleagues and jonas who was instrumental in providing again resources from pac bio for the sequencing and assembly and as you can see from this genome it's a relatively small genome expect it's one of the smallest among the butterflies or even the smallest if you if ing to adriana at 200 approximately 215 million bases i'm going to go rather quickly through these uh so as to save some time but the conteg n50 as you can see easily meets the ebp standard on the primary haplotype each of these assemblies was done with high fiasm and phased into primary and alternate haplotypes the uh the contagin 58.8 megabases and 7.3 megabase on the alternate really extraordinary as are the bosco scores with level of completeness 99 uh completeness complete genes in the primary assembly 95.2 percent in the alternate assembly second species i'm going to discuss is an endangered species of butterfly bears metal mark again this is a collaboration between uh uc davis genome center packed bio earth by a genome project as part of the pak bio earth biogenome uh pac bioware's earth biogenome project um consortium and the project is being led again by adriana and uh and jonas at pac bio another excellent assembly with an n50 contact size of 4.42 in the primary and 1.61 megabases in the alternate uh assembly completeness is really really good 97 8 97.8 on the primary as far as gene completeness and 83.9 percent in the alternate assembly next species is critically endangered the black abalone at one point in time the black abalone was the most abundant large marine mollusk on the west coast of north america but now because of overfishing and withering syndrome it has been much declined in its population size and iucn red list has classified the black abalone as critically endangered now chloe orland who is on your program you should be hearing from her sometime today is likely to discuss uh the black abalone and sequence so i won't take up much of her time but just to say again this is an amazing assembly fourteen point three megabase contig n50 for uh a genome size of just over one billion bases with very high uh busco scores in terms of gene completeness now moving to plants you've heard about two insects a mollusk now plants this is a different collaboration also done as part of the pac bio ebp program again a collaboration with uc davis genome center and our collaborators at the university uh at unicamp in brazil the university of campinas and ambrapa uh at the university of campinas and this is a a great improvement over the existing assembly there had been an assembly for this species but fellowsia are fascinating species uh among a group of of plants that exist in the camporepestry region of brazil these are sometimes called resurrection plants because they look like they're completely dead their leaves turn brown but the leaves aren't actually not dead as soon as the rains come these same leaves they don't die and fall off and newly regenerate they again turn green and they're called resurrection plants and this whole biological process is enormously interesting in under investigation by the group at uh imbrapa and unicamp and great assembly again exceeding the uh is a complicated genome with lots of repeats and other things which uh has so far interfered with creating an alternate assembly greater than one megabase but we've achieved 1.7 megabase contig and 50 for this genome and very high busco scores for both assemblies the last one i want to talk to you about is the large tree shrew and uh this one is uh being has been done in collaboration with oliver ryder at the san diego zoo who's provided uh the sample for this uh ollie and his staff at the frozen zoo and dovetail genomics and uh pac bio now this one is a completely scaffolded assembly some of the others are either in process or have been scaffolded i haven't shown you the data but this one has been scaffolded at uc davis uh the the lead investigator is a post-doc in my lab joanna damas and um the assembly has been done using the abbreviated version of the pipeline shown on the lower right side falcon unzip for the for the hifi data scaf10x for the uh for the the link reads and dovetail has done the high rise for all the data sets using high c and as you can see the scaffold n50 here is 7.4 megabase just for the hi-fi contigs and when you add in the scaffolding you've got a really high quality chromosome scale assembly at about 113 million bases for n50 important to also note here that the l90 which is the given set of contigs basically each of its own length uh you know the l90 is the count uh that is defined as the smallest number of contigs whose length is the sum that makes up 90 of genome size so 27 scaffolds make up 97 of the genome size which is very close to the number of chromosomes if you see in the upper right hand corner there you see that the um that the um that the chromosome number is 28 and so we're very excited about this particular uh assembly and what this assembly allows us to do is my my last data slide is um is fill in a missing part of the euthyrian tree so um we already have for our work on chromosome evolution and ancestral genome reconstructions at least a scaffold based to a chromosome level assembly for 18 of the 19 eutherian orders the scandanchia the species which is represented by uh the the order which is represented by the the large tree shrew is uh was was missing and uh it's a very interesting uh order because its phylogeny has not yet been resolved so having the sequence of this species will help us to understand where to place it's really the last group of organ is the last order that is not clearly resolved in the mammalian phylogeny so we'll be able to resolve that issue as well as uh provide a detailed reconstruction of the ancestral mammalian genomes representing all eutherian orders in most of the marsupial and monitoring orders as well okay so to wrap it up i'd close by returning to carolyn's conceptual slide which i think is useful we are now requiring genomes for endangered species at an extraordinary rate the ebp and its affiliated projects can serve as an important role in bridging the gap between genome scientists and conservation scientists and policy makers to give them better tools for decision making this is our responsibility to bridge this gap to make sure that those who need this information or who will use this information for the purposes of conservation have the information in a usable form that can help these programs to advance we expect that these programs will have a direct impact on conservation by slowing the rate of extinction of endangered species of plants and animals as well as other eukaryotes that underspin the biosphere so i want to close by thanking everyone go forth and sequence thank you very much and now i'll turn it back to you michelle for the rest of the program today thank you thank you so much harris for that wonderful talk on the ebp work you're doing such great work and i can't wait to see the progress as the years go by as a reminder we are hosting a live panel discussion at 12 15 pacific where we'll be discussing the future of sequencing for plant and animal sciences so i encourage you to join us for that in addition you will have the opportunity to take home a thank you gift for participating in this first ever pag bio day in order to get your gift simply go to one of these live sessions that i have listed here or attend more than one of the on-demand sessions all of the eligibility requirements will be determined on the back end you don't have to do anything beyond that and you can look out for an email from us to with instructions on how to get your take home gift and last but certainly not least we are giving away free hi-fi sequencing um with our smart grant program so if you have a collaborative research project that you're interested in doing i encourage you to go apply for our smart grant uh submit your proposal by the end of the day on january 22nd january 22nd which is coming up rather quickly but head to pacb.com smart grant for that and with that i just want to say thank you all so much for joining us on this exciting pag bio day and please feel free to get in touch with us directly at pacme.com scientist or check out our website