Comparative analysis of targeted long read sequencing approaches for characterization of a plant’s immune receptor repertoire

Abstract

BackgroundThe Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified.ResultsSequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn’t generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline.ConclusionThe described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3936-7) contains supplementary material, which is available to authorized users.

Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing

“…Read-lengths are limited only by input DNA lengths, and validated reads as long as 2,272,580 bases have been reported [2]. Nanopore sequencing has been used for various applications, including genome sequencing of Arabidopsis and a wild tomato relative [3, 4], resolving complex T-DNA insertions [5], and disease resistance gene enrichment sequencing [6].…”

ORF Capture-Seq: a versatile method for targeted identification of full-length isoforms

“…These approaches were initially developed for targeted sequencing of protein-coding regions of genomic DNA (whole exome sequencing) 13 and RNA fragments from short-read RNA-seq experiments (e.g., CaptureSeq) [14][15][16][17][18] . Such approaches have been adapted for targeted sequencing of long genomic fragments (>2kb) or full-length cDNA molecules [19][20][21][22][23][24] . In two notable studies described recently, complex pools of biotinylated oligos were used to enrich for and sequence thousands of protein-coding and lncRNA targets, leading to considerable gains in isoform detection and new insights about the nature of transcriptomic complexity 25,26 .…”

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

“…The Oxford Nanopore Technologies (ONT) MinION sequencer represents a significant paradigm shift in the reach, applicability, and capability of nucleic acid sequencing technology 1 . Combining a portable form factor, simple library prep, long-read capability (kb to Mb) 2 , direct RNA sequencing 3 , and real-time data output, the MinION has been variously applied to forensic genotyping 4 , bacterial typing 5 , plant biology 6 , food safety 7 , environmental metagenomics 8,9 , cancer research 10,11 , antibiotic resistance studies 12,13 , and de novo genome assembly [14][15][16] . The small operational and logistical footprint of the MinION, combined with its real-time capabilities 17 , make it uniquely suited to diagnostics and surveillance in clinical and field-forward settings, where the MinION has already been applied to assay Ebola 18,19 , Zika 20 , tuberculosis 21 , and other pathogens [22][23][24][25] .…”