I just got back from Great Lakes Bio 2017 (GLBIO2017) at the University of Illinois-Chicago (UIC) campus. It was a great meeting and I really enjoyed the quality of the research presented as well as the atmosphere of the campus and neighborhood.
I was very surprised by just how nice the Chicago “West Loop” neighborhood near Randolph Street and down towards Greektown really is. I had some great meals, including a memorable Italian dinner at Formentos.
But the purpose of this post is to briefly describe a few of my favorite talks from the meeting. So here goes, in no particular order:
Kevin White, Tempus Labs:
I was really impressed with Kevin White’s GLBIO2017 talk and demo of his company’s technology (despite the ongoing technical A/V issues!) Tempus labs is a clinical sequencing company but also an informatics company focused on cancer treatment that seeks to pull together all of the disparate pieces of patient data that float around in EHR databases and are oftentimes not connected in meaningful ways.
The company sequences patient samples (whole exome and whole genome) and then also hoovers up reams of patient EHR data using Optical Character Recognition (OCR), Natural Language Processing (NLP), and human expert curation to turn the free-form flat text of medical records from different clinics and systems into a form of “tidy data” that can be accessed from an internal database.
Then, clinical and genomic data are combined for each patient in a deep-learning system that looks at treatments and outcomes for other similar patients and presents the clinician with charts that show how patients in similar circumstances fared with varying treatments, given certain facts of genotype and tumor progression, etc… The system is pitched as “decision support” rather than artificial “decision making.” That is, a human doctor is still the primary decider of treatment for each patient, but the Tempus deep learning system will provide expert support and suggest probabilities for success at each critical care decision point.
The system also learns and identifies ongoing clinical trials, and will present relevant trials to the clinician so that patients can be informed of possibly beneficial trials that they can join.
Murat Eren, merenlab.org
Murat Eren’s talk on tracking microbial colonization in fecal microbiome transplantation (i.e., “poop pills”) was excellent and very exciting. Although the “n” was small (just 4 donors and 2 recipients) he showed some very interesting results from transferring fecal microbiota (FM) from healthy individuals to those with an inflammatory bowel disease.
Among the interesting results are the fact that he was able to assemble 97 metagenomes in the 4 donor samples. Following the recipients at 4 and 8-weeks post FM transplant showed that the microbial genomes could be classed into those that transfer and colonize permissively (both recipients), those that colonize one or the other recipient, and those that fail to colonize both. Taxa alone did not explain why some microbes colonized easily, while other failed to colonize.
He also showed that 8 weeks post FM transplant, the unhealthly recipients had improved symptoms but also showed that in a PCA analysis of the composition of the recipient gut and the healthy human gut from 151 human microbiome project (HMP) samples, the recipients moved into the “healthy” HMP cluster from being extreme outliers on day 0.
He also investigated differential gene function enrichment between the permissive colonizers and the microbes that never colonized recipient’s guts and found that sporulation genes may be a negative factor driving the failure (or success) of transplantation. He proposed that the recent and notable failure of the Seres microbiome drug in clinical trials may be owing to the fact that the company killed the live cultures in favor of more stable spore-forming strains when formulating the drug. His work would suggest that these strains are less successful at colonizing new hosts.
Bo Zhang, 3D genome browser
With the ever-increasing volume of genomic and regulatory data and the complexity of that data, there is a need for accessible interfaces to it. Bo Zhang’s group at Penn State has worked to make a new type of genome browser available that focuses on the 3D structure of the genome, pulling together disparate datatypes including chromatin interaction data, ChIP-Seq, RNA-Seq, etc… You can also browse a complete view of the regulatory landscape and 3D architecture of any region of the genome. You can also check the expression of any queried gene across hundreds of tissue/cell types measured by the ENCODE consortium. On the virtual 4C page, they provide multiple methods to link distal cis-regulatory elements with their potential target genes, including virtual 4C, ChIA-PET and cross-cell-type correlation of proximal and distal DHSs.
All in all, GLBIO2017 was a very enjoyable and informative meeting where I met a lot of great colleagues and learned much. I am looking forward to next year!
I am happy to say that myself and my collaborators in the Department of Occupational and Environmental Health here at the University of Iowa have had our recent work on the bacterial composition of poultry bioaerosols (i.e., the dust that poultry workers breath during their tasks) published in Microbial Biotechnology.
The key figure from this work is the following heat map that illustrates the top taxa that are common to all 21 samples:
What is remarkable about whole-genome shotgun metagenomics is that we are not only surveying bacterial DNA, but also viral, fungal, archaeal, and eukaryotic DNA in one experiment. You can see from the figure that certain viruses are found in all samples, but it is bacteria, particularly Lactobacillus and Salinicoccus, that are the most abundant.
Stay tuned because we will have a paper coming out soon on the fungal composition of these samples as well. In the case of this paper, and our next manuscript, it is the first time whole-genome shotgun metagenomics has been applied to the field of environmental health in poultry environments.
Intuitively, it is easy to grasp that the more sequencing depth (i.e., the greater the number of reads covering any given position in the genome) the more accurate the calling of SNPs and indels (insertions/deletions). But how much difference does this actually make in the real world? Is 20X coverage dramatically worse than 30X (considered a standard coverage depth on genomes)?
To find out, I conducted an experiment with the bcbio pipeline, a bioinformatics pipeline solution built in python that allows for automated and reproducible analyses on high-performance computing clusters. One feature of bcbio is that it can perform validation surveys using high-confidence consensus calls from reference genomes like the NA12878 Coriell sample (from the Genome in a Bottle project).
For NA12878, researchers collated consensus SNP and indel calls from a large variety of sequencing technologies and calling methods to produce a very high-confidence callset for training other methods or validating a sequencing workflow. bcbio includes these variant calls and can easily be setup to validate these calls against a sequenced NA12878 genome.
The sequencing depth experiment
I started with a NA12878 genome sequenced to 30X sequencing depth. To compare shallower depths, I subsampled the data to generate 20X, 10X, etc… [Please note: data was not subsetted randomly, rather “slices” were taken from the 30X dataset] To look at a 60X coverage datapoint, I combined data from two sequencing runs on both flow cells of a HiSeq4000 instrument.
The results after validation are shown in Figure 1 (depth of coverage is along the x-axis):
The figure shows that, as expected, when sequencing depth decreases the error rate increases, and SNP discovery declines. It also makes the case for the commonly held view that 30X is enough coverage for genomes, since going to 60X leads to almost unnoticeable improvement in the % found and a slight increase in error. Performance really degrades at 12X and below, with poor discovery rates and unacceptably high error rates.
I will be submitting a short manuscript to biorxiv.org soon describing this work in more detail.
According to a recent paper, the common practice of rarefying (randomly subsampling your 16S reads), is statistically incorrect and should be abandoned in favor of more sophisticated ‘mixture-model’ approaches favored by RNA-Seq analysis software.
The authors give a basic thought experiment to help clarify their reasoning. I’ve copied the figure below. It basically shows what happens when one rarifies library “B” from 1000 reads to 100 reads in order to match library “A.” This is a standard procedure, even in the QIIME workflow. By dividing the library size by 10, the variance of the data in B goes up by 10, and thus the statistical power to differentiate between OTU1 and OTU2’s abundances in samples A and B is lost.
As the authors point out in the paper:
“Although rarefying does equalize variances, it does so only by inflating the variances in all samples to the largest (worst) value among them at the cost of discriminating power (increased uncertainty). “
The solution to the rarefying problem is to take advantage of methods from the RNA-Seq world in order to determine differential taxonomic abundances, just as differential transcript abundances are calculated by the same methods. The R package, phyloseq, provides an R container for taxonomic datasets from Qiime (in the BIOM format). It also provides a convenient set of extensions that allow analysis of this data with RNA-Seq methods like edgeR and DESeq2.