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 2017

Spliceosome 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 2017

 

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