Improving genetic diagnosis in Mendelian disease with transcriptome sequencing, bioRxiv, 2016-09-09

AbstractExome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25-50%. Here, we explore the utility of transcriptome sequencing (RNA-seq) as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to over 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approaches.One Sentence SummaryTranscriptome sequencing improves the diagnostic rate for Mendelian disease in patients for whom genetic analysis has not returned a diagnosis.

biorxiv genomics 100-200-users 2016

In-field metagenome and 16S rRNA gene amplicon nanopore sequencing robustly characterize glacier microbiota, bioRxiv, 2016-09-08

ABSTRACTIn the field of observation, chance favours only the prepared mind (Pasteur). Impressive developments in genomics have led microbiology to its third “Golden Age”. However, conventional metagenomics strategies necessitate retrograde transfer of samples from extreme or remote environments for later analysis, rendering the powerful insights gained retrospective in nature, striking a contrast with Pasteur’s dictum. Here we implement highly portable USB-based nanopore DNA sequencing platforms coupled with field-adapted environmental DNA extraction, rapid sequence library generation and off-line analyses of shotgun metagenome and 16S ribosomal RNA gene amplicon profiles to characterize microbiota dwelling within cryoconite holes upon Svalbard glaciers, the Greenland Ice Sheet and the Austrian Alps. We show in-field nanopore sequencing of metagenomes captures taxonomic composition of supraglacial microbiota, while 16S rRNA Furthermore, comparison of nanopore data with prior 16S rRNA gene V1-V3 pyrosequencing from the same samples, demonstrates strong correlations between profiles obtained from nanopore sequencing and laboratory based sequencing approaches. gene amplicon sequencing resolves bacterial community responses to habitat changes. Finally, we demonstrate the fidelity and sensitivity of in-field sequencing by analysis of mock communities using field protocols. Ultimately, in-field sequencing potentiated by nanopore devices raises the prospect of enhanced agility in exploring Earth’s most remote microbiomes.

biorxiv microbiology 100-200-users 2016

 

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