Expressed Exome Capture Sequencing (EecSeq) a method for cost-effective exome sequencing for all organisms with or without genomic resources, bioRxiv, 2017-11-24
AbstractExome capture is an effective tool for surveying the genome for loci under selection. However, traditional methods require annotated genomic resources. Here, we present a method for creating cDNA probes from expressed mRNA, which are then used to enrich and capture genomic DNA for exon regions. This approach, called “EecSeq”, eliminates the need for costly probe design and synthesis. We tested EecSeq in the eastern oyster, Crassostrea virginica, using a controlled exposure experiment. Four adult oysters were heat shocked at 36° C for 1 hour along with four control oysters kept at 14° C. Stranded mRNA libraries were prepared for two individuals from each treatment and pooled. Half of the combined library was used for probe synthesis and half was sequenced to evaluate capture efficiency. Genomic DNA was extracted from all individuals, enriched via captured probes, and sequenced directly. We found that EecSeq had an average capture sensitivity of 86.8% across all known exons and had over 99.4% sensitivity for exons with detectable levels of expression in the mRNA library. For all mapped reads, over 47.9% mapped to exons and 37.0% mapped to expressed targets, which is similar to previously published exon capture studies. EecSeq displayed relatively even coverage within exons (i.e. minor “edge effects”) and even coverage across exon GC content. We discovered 5,951 SNPs with a minimum average coverage of 80X, with 3,508 SNPs appearing in exonic regions. We show that EecSeq provides comparable, if not superior, specificity and capture efficiency compared to costly, traditional methods.
biorxiv genomics 0-100-users 2017Computational haplotype recovery and long-read validation identifies novel isoforms of industrially relevant enzymes from natural microbial communities, bioRxiv, 2017-11-23
AbstractPopulation-level diversity of natural microbiomes represent a biotechnological resource for biomining, biorefining and synthetic biology but requires the recovery of the exact DNA sequence (or “haplotype”) of the genes and genomes of every individual present. Computational haplotype reconstruction is extremely difficult, complicated by environmental sequencing data (metagenomics). Current approaches cannot choose between alternative haplotype reconstructions and fail to provide biological evidence of correct predictions. To overcome this, we present Hansel and Gretel a novel probabilistic framework that reconstructs the most likely haplotypes from complex microbiomes, is robust to sequencing error and uses all available evidence from aligned reads, without altering or discarding observed variation. We provide the first formalisation of this problem and propose “metahaplome” as a definition for the set of haplotypes for any genomic region of interest within a metagenomic dataset. Finally, we demonstrate using long-read sequencing, biological evidence of novel haplotypes of industrially important enzymes computationally predicted from a natural microbiome.
biorxiv bioinformatics 0-100-users 2017New synthetic-diploid benchmark for accurate variant calling evaluation, bioRxiv, 2017-11-23
Constructed from the consensus of multiple variant callers based on short-read data, existing benchmark datasets for evaluating variant calling accuracy are biased toward easy regions accessible by known algorithms. We derived a new benchmark dataset from the de novo PacBio assemblies of two human cell lines that are homozygous across the whole genome. This benchmark provides a more accurate and less biased estimate of the error rate of small variant calls in a realistic context.
biorxiv bioinformatics 100-200-users 2017Recovery of gene haplotypes from a metagenome, bioRxiv, 2017-11-23
AbstractElucidation of population-level diversity of microbiomes is a significant step towards a complete understanding of the evolutionary, ecological and functional importance of microbial communities. Characterizing this diversity requires the recovery of the exact DNA sequence (haplotype) of each gene isoform from every individual present in the community. To address this, we present Hansel and Gretel a freely-available data structure and algorithm, providing a software package that reconstructs the most likely haplotypes from metagenomes. We demonstrate recovery of haplotypes from short-read Illumina data for a bovine rumen microbiome, and verify our predictions are 100% accurate with long-read PacBio CCS sequencing. We show that Gretel’s haplotypes can be analyzed to determine a significant difference in mutation rates between core and accessory gene families in an ovine rumen microbiome. All tools, documentation and data for evaluation are open source and available via our repository <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsgithub.comsamstudio8gretel>httpsgithub.comsamstudio8gretel<jatsext-link>
biorxiv bioinformatics 100-200-users 2017Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution, bioRxiv, 2017-11-23
SummaryTools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non-canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression.Highlights<jatslist list-type=bullet><jatslist-item>Measurement of spliceosome-bound precursor and intermediate in three species<jatslist-item><jatslist-item>Non-canonical splicing events revealed<jatslist-item><jatslist-item>Statistical modeling uncovers substrate features that predict catalytic efficiency<jatslist-item><jatslist-item>Discard of suboptimal substrates occurs in vivo and predicts intron-retained mRNAs<jatslist-item>
biorxiv molecular-biology 0-100-users 2017Current CRISPR gene drive systems are likely to be highly invasive in wild populations, bioRxiv, 2017-11-21
AbstractRecent reports have suggested that CRISPR-based gene drives are unlikely to invade wild populations due to drive-resistant alleles that prevent cutting. Here we develop mathematical models based on existing empirical data to explicitly test this assumption. We show that although resistance prevents drive systems from spreading to fixation in large populations, even the least effective systems reported to date are highly invasive. Releasing a small number of organisms often causes invasion of the local population, followed by invasion of additional populations connected by very low gene flow rates. Examining the effects of mitigating factors including standing variation, inbreeding, and family size revealed that none of these prevent invasion in realistic scenarios. Highly effective drive systems are predicted to be even more invasive. Contrary to the National Academies report on gene drive, our results suggest that standard drive systems should not be developed nor field-tested in regions harboring the host organism.
biorxiv synthetic-biology 100-200-users 2017