September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

September 22, 2019  |  

NextSV: a meta-caller for structural variants from low-coverage long-read sequencing data.

Structural variants (SVs) in human genomes are implicated in a variety of human diseases. Long-read sequencing delivers much longer read lengths than short-read sequencing and may greatly improve SV detection. However, due to the relatively high cost of long-read sequencing, it is unclear what coverage is needed and how to optimally use the aligners and SV callers.In this study, we developed NextSV, a meta-caller to perform SV calling from low coverage long-read sequencing data. NextSV integrates three aligners and three SV callers and generates two integrated call sets (sensitive/stringent) for different analysis purposes. We evaluated SV calling performance of NextSV under different PacBio coverages on two personal genomes, NA12878 and HX1. Our results showed that, compared with running any single SV caller, NextSV stringent call set had higher precision and balanced accuracy (F1 score) while NextSV sensitive call set had a higher recall. At 10X coverage, the recall of NextSV sensitive call set was 93.5 to 94.1% for deletions and 87.9 to 93.2% for insertions, indicating that ~10X coverage might be an optimal coverage to use in practice, considering the balance between the sequencing costs and the recall rates. We further evaluated the Mendelian errors on an Ashkenazi Jewish trio dataset.Our results provide useful guidelines for SV detection from low coverage whole-genome PacBio data and we expect that NextSV will facilitate the analysis of SVs on long-read sequencing data.

September 21, 2019  |  

Long-read genome sequencing identifies causal structural variation in a Mendelian disease.

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions?>?50?bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184?bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.

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