Bird, S. W. | Search bioRxiv

bioRxiv2018

Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria

The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-by-molecule real-time selective sequencing or "Read Until". As DNA transits a MinION nanopore, real-time pore current data can be accessed and analyzed to provide active feedback to that pore. Fragments of interest are sequenced by default, while DNA deemed non-informative is rejected by reversing the pore bias to eject the strand, providing a novel means of background depletion and/or target enrichment. In contrast to the previously published pattern-matching Read Until approach, our RUBRIC method is the first example of real-time selective sequencing where on-line basecalling enables alignment against conventional nucleic acid references to provide the basis for sequence/reject decisions. We evaluate RUBRIC performance across a range of optimizable parameters, apply it to mixed human/bacteria and CRISPR/Cas9-cut samples, and present a generalized model for estimating real-time selection performance as a function of sample composition and computing configuration.

bioinformatics↗

bioRxiv2017

A new sequencing-based women’s health assay combining self-sampling, HPV detection and genotyping, STI detection, and vaginal microbiome analysis

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a womans reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive womens health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 32 bacterial taxa of clinical importance, including Lactobacillus, Sneathia, Gardnerella, and 4 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, healthy ranges were determined in a group of 50 self-reported healthy women. The hrHPV portion of the test was evaluated against the Digene High-Risk HPV HC2 DNA test with vaginal samples obtained from 185 women. Results were concordant for 181/185 of the samples (overall agreement of 97.83%, Cohens kappa = 0.93), with sensitivity and specificity values of 94.74% and 98.64%, respectively. Two discrepancies were caused by the Digene assays known cross-reactivity with low-risk HPV types, while two additional samples were found to contain hrHPV not detected by Digene. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

microbiology↗