ISSRseq: An extensible method for reduced representation sequencing

Abstract

The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of biology, including crop breeding, pathology, forensics, forestry, ecology, evolution and conservation. However, the wet‐laboratory expertise and bioinformatics training required to conduct genome‐scale variant discovery remain limiting factors for investigators with limited resources. Here we present ISSRseq, a PCR‐based method for reduced representation of genomic variation using simple sequence repeats as priming sites to sequence inter simple sequence repeat (ISSR) regions. Briefly, ISSR regions are amplified with single primers, pooled, used to construct sequencing libraries with a commercially available kit, and sequenced on the Illumina platform. We also present a flexible bioinformatic pipeline that assembles ISSR loci, calls and hard filters variants, outputs data matrices in common formats, and conducts population analyses using R. Using three angiosperm species as case studies, we demonstrate that ISSRseq is highly repeatable, necessitates only simple wet‐laboratory skills and commonplace instrumentation, is flexible in terms of the number of single primers used, and can generate genomic‐scale variant discovery on par with existing RRS methods which require more complex wet‐laboratory procedures. ISSRseq represents a straightforward approach to SNP genotyping in any organism, and we predict that this method will be particularly useful for those studying population genomics and phylogeography of non‐model organisms. Furthermore, the ease of ISSRseq relative to other RRS methods should prove useful to those lacking advanced expertise in wet‐laboratory methods or bioinformatics.

Digitized collections elucidate invasion history and patterns of awn polymorphism inMicrostegium vimineum

“…Barcoded read sets for each individual were run through a custom pipeline (https://github.com/btsinn/ISSRseq), which comprises a set of UNIX bash scripts to assemble a pseudoreference, clean and map reads to the reference, and call variants following Sinn et al (2021). The resulting filtered .vcf file was used in downstream analyses.…”

“…We used the CTAB method for DNA extraction (Doyle and Doyle, 1987) from 0.2 g silica-dried leaf tissue, one individual serving as a voucher from each locality. We used a recently developed protocol based on sequencing of inter-simple sequence repeat amplicons (ISSR-seq; Sinn et al, 2021). Briefly, gDNAs were quantified via Qubit Broad Range dsDNA assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and diluted to 20 ng/µL for amplification using four ISSR motifs in multiplex from UBC set no.…”

“…9 primers 848 [(CA) 8 RG], 857 [(AC) 8 YG], 868 [(GAA) 6 ], and 873 [(GACA) 4 ]. PCR reactions, library preparation, and Illumina NextSeq2000 sequencing (v3 chemistry, 2 × 100 bp paired end reads) were conducted as in Sinn et al (2021).…”

Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping

“…In recent years, the rapid development of next-generation sequencing (NGS) technology has made it possible to rapidly identify large numbers of polymorphisms in genomes. To reduce genome complexity, several NGS polymorphism-detection methods have been developed that do not require the entire genome to be read, including restriction site-associated DNA sequencing (RAD-seq), genotyping by sequence (GBS), double-digest RAD-seq (ddRAD-seq), multiplexed inter-simple sequence repeat (ISSR) genotyping by sequencing (MIG-seq), and ISSR sequencing (Baird et al ., 2008; Elshire et al ., 2011; Peterson et al ., 2012; Sinn et al ., 2022; Suyama et al ., 2021; Suyama and Matsuki, 2015) (Fig. 1).…”

“…Using MIG-seq, a higher number of polymorphisms can be obtained for species with large genomes (Nishimura et al ., 2022). ISSR sequencing is a method used to amplify the ISSR region via PCR and is comparable to GBS and RAD-seq in terms of polymorphism detection; however, ISSR sequencing requires an additional fragmentation step during library construction and is more time-consuming than MIG-seq (Sinn et al ., 2022).…”