AGBT 2015 Summary of Day 3

Advances in Genome Biology and Technology Conference 2015

Day 3 of the Advances in Genome Biology and Technology meeting in Marco Island began with an announcement that next year the meeting would be held in Orlando due to hotel renovations, eliciting a groan from the audience. The meeting will come back to Marco Island in 2017.

Today’s plenary session speakers all presented work with a clinical focus, acknowledgement by the conference organizers about the direction of genome sequencing. The first speaker, Gail Jarvik, head of medical genetics at the University of Washington Medical Center presented on lessons learned from the Clinical Sequencing Exploratory Research (CSER) Consortium, marketed as ‘Hail CSER’. CSER is a national consortium of projects aimed at sharing innovations and best practices in the integration of genomic sequencing into clinical care. CSER has established a list of 112 actionable genes, some overlapping with the American College of Medical Genetics (ACMG) list. The CSER group annotated pathogenic and novel variants of the Exome Variant Server (EVS) to estimate rates in individuals of European and African ancestry.

The next talk was by Euan Ashley on moving toward clinical grade whole genome sequencing. He started by describing the genome as complex, full of repeats, duplications and paralogous sequences, giving him ‘a cold sweat at night’. He gave an example of a study with 12 adult participants who underwent WGS and described how clinical grade sequencing demands consistency in reporting. Most variants annotated as pathogenic were downgraded after manual review, but this takes lots of time. 12 individuals with 1,000 variants took around 1 hour per variant. In this case the use of WGS was associated with incomplete coverage of inherited disease genes, low reproducibility of detectable genetic variation and uncertainty about clinically reportable findings. He commented that new algorithms would be needed to address these problems and that ‘we’re at the beginning of genomics medicine’. Parts of his talk can be seen in his presentation at PMWC last month.

The last presentation before the break was by Levi Garraway who discussed the goal of cancer precision medicine to develop new therapeutics and combinations against molecular defined tumors. He mentioned that there are many discovery opportunities in clinical cancer genomics especially in terms of response and resistance to new therapies. Garraway sequenced the genomes of 57 prostate tumors and matched normal tissues to study somatic alterations. His model suggests that chormoplexy induces considerable genomic derangement over a relatively few number of events in prostate cancer supporting a model of punctuated cancer evolution. He introduced a 10X Genomics approach for phasing of large – 100 kb regions with exonic baits to obtain rearrangement information for chromoplexy. In the end he emphasized the importance of RNA-Seq profiling in conjunction with DNA sequencing for translational medicine to be relevant.

After the break, Stephen Kingsmore gave a presentation on rapid genome sequencing for genetic disease diagnostics in neonatal intensive care units. Kingsmore began the talk by describing how newborn screening (NBS) and early diagnosis reduces morbidity and mortality. NGS of 60 genetic diseases identifies ~5,000 affected newborns each year. He described how rapid genome sequencing (RGS) has the potential to improve NBS to most genetic diseases in newborns admitted to level II-IV NICUs. He mentioned a ‘ultra rapid’ sequencing pipeline he developed along with Illumina that takes 28 hours to go from sample to variant annotation (not publically available). He also discussed NSIGHT, a consortium for newborn sequencing sponsored by the NIH to understand the role of genome sequencing. More details can be found on the NHGRI page.

The last two plenary talks were by Christian Matranga and Malachi Griffith. Matranga described the clinical sequencing of viral genomes as important to understanding the evolution and transmission of the pathogen and the ability to inform on surveillance and therapeutic development. They developed a sequencing approach that combines RNAse H based depletion of rRNA with random primed cDNA RNA-seq to detect and assemble genomes from divergent lineages. They sequenced ~300 Lassa (LASV) and ~100 Ebola (EBOV) genomes. We describe some of their efforts in an earlier post called, Sequencing Suggests the Ebola Virus Genome is Changing. Be sure to read the New Yorker reference, it’s compelling!

Griffith’s talk was on optimizing genome sequencing and analysis. He makes the point that while most tumors are sequenced by exome sequencing at 75-100x mean coverage or by whole genome sequencing (WGS) to 30-50x mean coverage, detection of low frequency mutations require greater depth. He performed deep sequencing of an acute myeloid leukemia (AML) by WGS up to 350X, whole exome to 300X and using a capture panel of ~260 recurrently mutated AML genes to ~10,000x coverage. He found that deeper sequencing revealed more driver variants and improved the assignment of variants to clonal clusters. Checkout his animation of WGS depth down-sampling.

After lunch began the ‘Bronze sponsor workshops’, essentially the talks you pay >$40K to give. The most interesting was the last by 10X Genomics, mainly because as @bioinformer put it, “10X Genomics is the new princess of the AGBT ball”. First, check out the video that received a round of applause from the AGBT crowd: Changing the Definition of Sequencing. They announced their instrument would be available in Q2 this year, cost ~$75K and $500 / sample. This brings the question whether 10X Genomic’s microfluidic platform offers greater potential than Molecule. What are the implications for Illumina or PacBio? To learn more check out Keith Robison’s insightful post detailing all there is currently known about 10X Genomics.

After dinner began concurrent sessions on technology, genomic medicine and transcriptomics. Hopefully someone else will post details about the genomic medicine and transcriptomics sessions. The technology session began with Iain Macaulay describing G&T-seq, separation and parallel sequencing of genomes and transcriptomes of single cells. This was the first talk this year at AGBT with an embargo, no tweets were allowed. So rather than go into details, we did find this lecture online. The next talk was by Alexandre Melnikov on MITE-Seq, an approach to site directed mutagenesis referred to as Mutagenesis by Integrated TiLEs. MITE facilitates structure-function studies of proteins at higher resolution than typical site directed approaches. To read more check out their paper published last year in Nucleic Acid Research. Andrea Kohn, then described single-cell methylome profiling of Aplysia neurons. Using methyl-dip and bisulfite sequencing she achieved >20x coverage for each neuron and then added RNA-seq providing the first methylome and transcriptome from a single neuron. Next up was Sara Goodwin who gave an in depth analysis of the Oxford MinION Device for de novo and cDNA sequencing. She sequenced the yeast strain W303 to over 120x coverage and was able to achieve up to 80% aligned reads. She mentioned that identifying the right aligner was still a work in progress but overall found promise in the technology for long read sequencing, de novo assembly and splice site id.

Tomorrow’s plenary talks are the second installment of genomics, ‘Genomics II’ with presentations by Michael Fischback, Rob Knight, Chris Mason, and Gene Myers, excellent lineup to close the final day of AGBT. Checkout our earlier posts if you’ve missed day 1 or day 2

AGBT 2015 Summary of Day 2

Advances in Genome Biology and Technology Conference


Day 2 of the Advances in Genome Biology and Technology Conference (AGBT 2015) began with high expectations as plenary speakers in day 1 set the bar high with engaging lectures.  The first talk in the morning was by Evan Eichler who described human genetic variation by single molecule sequencing. He generated 40x sequence coverage of a haploid human genome with average read lengths of 9 kbp. His method allows for the detection of indel and structural variants from several bases up to 20 kbp. Comparing his single haplotype to the human reference he resolved some ~26K indels at SVs at the basepair level. His analysis found complex variations such as mobile element insertions as well as inversions. His results suggest systematic bias against longer, complex repetitive regions can now be resolved. According to Eichler, ‘we need to capture SVs to make precision medicine, precision’. 

Elaine Mardis spoke next giving a super lecture comprised of four very interesting vignettes on advances in cancer genomics: markers of late relapse estrogen receptor disease, translating AML and translating cancer genomes into clinical care. She spoke of the database of curated mutations (DoCM), curated database of known disease-causing mutations that provide explorable variant lists and direct links to source citations. She also mentioned the opening of Clinical Interpretations of Variants in Cancer (CIVIC) for crowdsourcing later this year.

Next, Nuria Lopez-Bigas spoke about analyzing thousands of tumor genomes to identify cancer drivers and targeted therapeutic opportunities. She claimed that 90% of tumors have at least one driver mutation, with differences between tumor types. Some of these tumor types like melanoma have many drug opportunities while others like renal carcinoma have few. 36% of patients could benefit from targeted drugs for more than one driver. She described the release of a beta version of Integrative Onco Genomics, Intogen that uses mutations from several sources: tumor mutation data from ICGC, driver id methods from OncodriveCLUST, and mutation mapping by Ensembl VEP.

Substituting for Meredith Carpenter, Carlos Bustamante talked about PhenoCap, a targeted capture panel for comprehensive phenotyping of forensic DNA samples. PhenoCap allows the prediction of phenotypic traits ranging from autosomal ancestry to facial morphology. Bustamante’s group aims to make PhenoCap analogous to a forensic exome that provides a comprehensive profile for all sample types.  General utility of forensic DNA is hampered by heavily degraded and contaminated samples making traditional STR and PCR approaches unreliable, PhenoCap solves this. Another part of his talk focused on the eastern Indian slave trade, specifically sequencing of DNA from a slave cemetery in Mauritius and the broad diversity of ancestry discovered.

The last talk before lunch was by Eric Green from NHGRI on the new ‘US Precision Medicine Initiative’ (PMI). He thanked the organizers for keeping a place for him despite his reluctance to give a title or description of what he wanted to talk about, it was before PMI was even announced at the State of the Union Speech. He started by emphasizing President Obama’s longstanding interest in genomic medicine. As a senator he tried to pass a bill called the Genomics and Personalized Medicine Act in 2006. What was announced was $200M for FY16, $70M for cancer studies near term and $130M for long term cohort work, sequencing of 1 million volunteers. What doesn’t add up are the costs to sequence 1M people and the budget allocated for this. 30x WGS: $1,750 x 1M people = ~$2B, will there be more funds for this initiative? $200M falls short. Checkout Francis Collins (left most, pointing to the TV) watching the State of the Union at his home.  He wasn’t sure exactly what President Obama would say.

Francis Collins watching state of union speech

Sarah Tishkoff gave the next talk on adaptive traits in Africa. A nice summary of this work can be found here: Genetic Variation and Adaptation in Africa: Implications for Human Evolution and Disease. Her findings on GC biased gene conversion and its importance in population genetics is described in a recent paper: Biased Gene Conversion Skews Allele Frequencies in Human Populations, Increasing the Disease Burden of Recessive Alleles.

David Page next spoke on what single haplotype iterative mapping and sequencing (SHIMS) tells us about sex chromosomes. He started by describing all genes that are involved in sperm production as palindromic copies on the Y chromosome, the longest being 3Mbp. The male specific region of the Y chromosome (MSY) sequence reveals lineage- specific convergent acquisition and amplification of X-Y gene families, potentially fueled by a fight between acquired X-Y homologs. He also spoke of the goal to make reference grade sequencing of structurally complex regions faster and more affordable. His work, Sequencing the Mouse Y Chromosome Reveals Convergent Gene Acquisition and Amplification on Both Sex Chromosomes was recently published in Cell late last year.

Then next plenary talk in evolutionary genomics was by Erich Jarvis on the challenges of sequencing representative genomes for an entire vertebrate lineage. As part of the Avian Phylogenomics Consortium (>200 researchres, >120 institutions), he collected and sequenced genomes of 48 avian species representing nearly all orders. He pinpointed the rise of modern birds from mass extinctions and described the convergent expression of 55 genes in birds and human neurological regions as related to vocal learning. Much of this work is described in 8 articles in a special issue of Science called, A Flock of Genomes.

The next last two talks of the plenary session were by Jessica Alfoldi and James Bradner. Jessica’s presentation, Evolution Two Ways: Natural And Artificial Selection showed that changes in allele frequencies of non-coding ancestral variation allows for bursts of speciation in cichlids and behavior changes in rabbits, both on short time scales.  Bradner’s talk was on the  disruption of ‘super enhancers’.

The concurrent sessions began in the evening with topics in biology, informatics and cancer. Hopefully someone else will write about the informatics and cancer sessions. The biology session began with Hie Lim Kim describing WGS of five Nambian Khoisan individuals and a  population analysis study using a 420K SNP dataset to show that two genomes contained exclusive Khoisan ancestry. Climate data along with sequencing indicates an ancient split in modern humans. Karyn Meltz Steinberg’s talk was on exome sequencing of 20,000 Finns. The ability to detect genetic associations with Finns is improved due to a bottleneck that resulted in enrichment of low frequency, deleterious variants. Using three different analysis pipelines they generated a genotype consensus call set and identified several potentially novel associations with plasma adiponectin and plasma CRP levels. Charlotte Lindqvist presented a genome scale study of population dynamics and speciation in polar and brown bears. She found evidence for functional speciation related to cellular respiration, hibernation and pigmentation. Beth Shapiro presented her work on brown and polar bear interspecies hybridization, Matthew Blow talked about sequencing based approaches for genome scale functional annotation and Max Seibold presented on integrated RNA-seq of the host and microbe in the nasal airway of childhood asthmatics.

Tomorrow the talks will clinical based, with concurrent sessions in technology, genomic medicine and transcriptomics. If you missed the first day at AGBT, check our summary of day 1.  

AGBT 2015 – Summary of Day 1

AGBT 2015

Highlights of Day 1 at AGBT

‘Welcome to paradise’, first words by Rick Wilson kicking off the annual Advances in Genome Biology and Technology (AGBT) meeting.  The plenary session began with David Goldstein from Columbia University presenting, “Toward Precision Medicine in Neurological Disease”.  David’s talk began with a discussion of clinical sequencing for neurological diseases, specifically large scale gene discoveries in epileptic encephalopathies. In epilepsy, 12% of patients are ‘genetically explained’ by a casual de novo mutation, which allows for the application of precision medicine. He discussed how a K+ channel plays a key role in at least two different epilepsies and how Quinidine which has never been used for epilepsy, was being used as a targeted treatment. He cautioned that in the literature there are too may correlation studies that don’t really amount to much and as we use genetics to target diseases, it’s critical to perform proper genetics driven precision medicine and not put patients on wrong treatment plans. To better characterize the effects of mutations, he emphasized the need for solid model systems. He also mentioned that he believes truly complex diseases can be tackled with enough patients, numbers matter. To illustrate his point, he described the sequencing of over 3,000 ALS patients to get a clear picture of what genes/proteins have therapeutic importance. At the end of his talk he was asked the old whole genome sequencing (WGS) vs. whole exome sequencing (WES) question and replied that WES was sufficient, as WGS added little due to lack of interpretability. This touched off some debate in the audience and Twitter with regards to Exome-seq and WGS. Highlighted are the advantages of each approach here:

The second talk in the plenary session was by Richard Lifton, from Yale and it was titled, “Genes, Genomes and the Future of Medicine”. Richard cautioned the audience, describing the rush to sequence whole genomes as more industry driven than good science, essentially reiterating the point that WGS is hard to interpret. This began a side discussion on Twitter about those who agree and disagree with this sentiment. Most notably, Gholson Lyon referenced two recent papers that demonstrated new ways to make processing of WGS data easier: and that the accuracy of INDEL detection was greater in WGS compared to WES, even in targeted regions. On the cost front, WGS at 35x coverage currently costs $1,750: or $3,500 for 70X coverage, while whole exome sequencing costs at 100x coverage are around $1,314:  Richard remarked at the end of his talk that not much had been found in non-coding regions, several in the audience challenged him on this assessment.

The third talk in the plenary session was by Yaniv Erlich titled, “Dissecting the Genetic Architecture of Longevity Using Massive-Scale Crowd Sourced Genealogy”. We’ve had the pleasure to hear Yaniv give several lectures in the past, all have been engaging, this was no different. His talk was on using social media to dissect the genetic architecture of complex traits, specifically whether they work independently (additive) or together (epistatic). Predictions of epistasis suggest an exponential increase with added risk alleles. He used to dissect complex traits in large family trees and validated publicly submitted trees using genetic markers. He encoded birthplace as GPS coordinates using Yahoo Maps and showed migration from the Middle Ages through the early 20th Century. The video he played was amazing, check it out: His take home message was that longevity is an additive trait, which is good for personalized medicine. The Geni data he described is open to the public for use.

The fourth and final talk of the night was by Steven McCarroll titled, “A Common Pre-Malignant State, Detectable by Sequencing Blood DNA”. He started by posing the questions, what happens in the years before a disease becomes apparent; cancer genomes are usually studied when there are enough mutations to drive malignancy, do they happen in a particular order? He examined 12,000 exomes for somatic variants at low allele frequency and uncovered 3,111 mutations. Blood derived schizophrenia clustered in four genes: DNMT3A, TET2, ASXL1 and PPM1D, all disruptive. He postulates that driver mutations give cells an advantage, over several years clonal progeny takes over creating pre-cancerous cells. Therefore clonal hematopoiesis with somatic mutations can be readily detected by DNA sequencing and should become more common as we age. Patients with clonal mutations have a 12 fold higher rate of blood cancer, meaning there is a window for early detection, possibly 3 years. This work was recently published in the New England Journal of Medicine: Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.

Today’s sessions were impressive and set expectations high for tomorrow’s talks. McCarroll’s last comment nicely captured this sentiment, setting the tone for the rest of the meeting, “Medicine thinks of health and illness, there is a lot in between that is ascertainable via genome sequencing”. 

Whole Genome Sequencing (WGS) vs. Whole Exome Sequencing (WES)

Gene, exon, intron, sequencing


“Should I choose whole genome sequencing (WGS) or whole exome sequencing (WES) for my project?” is such a frequently posed question during consultation on Genohub, we thought it would be useful to address it here. With unlimited resources and time, WGS is a clear winner as it allows you to interrogate single-nucleotide variants (SNVs), indels, structural variants (SVs) and copy number variants (CNVs) in both the ~1% part of the genome that encodes protein sequences and the ~99% of remaining non-coding sequences. WES still costs a lot less than WGS, allowing researchers to increase sample number, an important factor for large population studies. WES does however have its limitations. Below we’ve highlighted the advantages of WGS vs. WES and described a real case example of someone ordering these services using Genohub.

Advantages of Whole Genome Sequencing

  1. Allows examination of SNVs, indels, SV and CNVs in coding and non-coding regions of the genome. WES omits regulatory regions such as promoters and enhancers.
  2. WGS has more reliable sequence coverage. Differences in the hybridization efficiency of WES capture probes can result in regions of the genome with little or no coverage.
  3. Coverage uniformity with WGS is superior to WES. Regions of the genome with low sequence complexity restrict the ability to design useful WES capture baits, resulting in off target capture effects.
  4. PCR amplification isn’t required during library preparation reducing the potential of GC bias. WES frequently requires PCR amplification as the bulk input amount needed to capture is generally ~1 ug of DNA.
  5. Sequencing read length isn’t a limitation with WGS. Most target probes for exome-seq are designed to be less than 120 nt long, making it meaningless to sequence using a greater read length.
  6. A lower average read depth is required to achieve the same breath of coverage as WES.
  7. WGS doesn’t suffer from reference bias. WES capture probes tend to preferentially enrich reference alleles at heterozygous sites producing false negative SNV calls.
  8. WGS is more universal. If you’re sequencing a species other than human your choices for exome sequencing are pretty limited.

Advantages of Whole Exome Sequencing

  1. WES is targeted to protein coding regions, so reads represent less than 2% of the genome. This reduces the cost to sequence a targeted region at a high depth and reduces storage and analysis costs.
  2. Reduced costs make it feasible to increase the number of samples to be sequenced, enabling large population based comparisons.

Most functional related disease variants can be detected at a depth of between 100-120x (1) which definitely makes the cost case for exome sequencing. Today on Genohub if you want to perform whole human genome sequencing at a depth of ~35X, the cost is roughly $1700/sample. If you were to request human exome-sequencing services with 100x coverage, using a 62 Mb target region, your cost would be $550/sample. Both of these prices include library preparation. So in terms of producing data WES is still significantly cheaper than WGS. It’s important to note that this doesn’t include your data storage and analysis costs which can also be quite a bit higher with whole genome sequencing.

It’s also important to remember that depth isn’t everything. The better your uniformity of reads and breath of coverage, the higher the likelihood you’ll actually find de novo mutations and call them. And that’s the main goal, if you can’t call SNPs or INDELs with high sensitivity and accuracy, then the most high depth sequencing runs are worthless.

To conclude, whole genome sequencing typically offers better uniformity and balanced allele ratio calls. While greater exome-seq depth can match this, sufficient mapped depth or variant detection in specific regions may never reach the quality of WGS due to probe design failures or protocol shortcomings. These are important considerations when examining tissues like primary tumors where copy number changes and heterogeneity are confounding factors.

If you’re ready to start an exome project, spend a few minutes determining the coverage you’ll need for your experiment. We have an exome-seq guide with examples to help you determine the number of sequencing reads you need to achieve a certain coverage of your exome. If you’re planning to embark on whole genome sequencing, use our NGS Matching Engine which automatically calculates the amount of sequencing capacity on various platforms to meet the coverage requirements for your project.


1) Effect of Next-Generation Exome Sequencing Depth for Discovery of Diagnostic Variants