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

Abstract

The Oxford MinION, the first commercial nanopore sequencer, is also the first to implement molecule-bymolecule 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.

Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED

“…The even/odd run used a single flowcell, where UNCALLED only monitored the even numbered channels and the odd channels were used as a control. This type of control has been used in previous ReadUntil applications 11 , 14 . Once each run finished, all reads were basecalled with guppy and mapped to a reference containing all Zymo genomes with minimap2 in order to classify reads as originating from yeast or bacteria ( Fig.…”

“…However, this method does not scale to references larger than tens of kilobases as the runtime is quadratic in the length of the sequence 11 . Others have attempted basecalling followed by mapping with a DNA aligner 14 , 15 , but basecalling is computationally expensive, and most basecallers are designed to work with fully sequenced reads and require a sizable amount of input signal to output a sequence. A ReadUntil method should ideally be fully streaming, meaning it can continuously refine its classification as more signal is produced.…”

A lab in the field: applications of real-time, in situ metagenomic sequencing

“…Later, Edwards et al . [ 103 ] showed the ability of Read Until strategies to enrich E. coli genomic sequences over human DNA. However, the actual revolution in targeted ONT sequencing is taking place in the recent months, with three different approaches being simultaneously released ( Table 3 ).…”

Genomics of host-pathogen interactions: challenges and opportunities across ecological and spatiotemporal scales

“…After DNA extraction, selective whole-genome amplification can specifically enrich for pathogen sequences in various ways: (i) by using oligos that are more abundant in the pathogen genome (Melnikov et al, 2011); (ii) by targeting differences in methylation between host and pathogen genomes (Gómez-Díaz et al, 2012); (iii) by sequence capture enrichment protocols for pathogen DNA (Tagle et al, 1993); or (iv) by Nanopore Cas9-targeted sequencing, a selective ligation of sequence adaptors at cut sites of Cas9 (Gilpatrick et al, 2019). Lastly, real-time evaluation of sequence data such as is possible with Nanopore technology could be further exploited to enrich for pathogen sequences during active sequencing (Edwards et al, 2019). When enrichment protocols are not feasible, host and pathogen associated reads can often be separated in silico using reference sequence databases.…”