Stacks 2: Analytical methods for paired‐end sequencing improve RADseq‐based population genomics

Abstract

For half a century population genetics studies have put type II restriction endonucleases to work. Now, coupled with massively‐parallel, short‐read sequencing, the family of RAD protocols that wields these enzymes has generated vast genetic knowledge from the natural world. Here, we describe the first software natively capable of using paired‐end sequencing to derive short contigs from de novo RAD data. Stacks version 2 employs a de Bruijn graph assembler to build and connect contigs from forward and reverse reads for each de novo RAD locus, which it then uses as a reference for read alignments. The new architecture allows all the individuals in a metapopulation to be considered at the same time as each RAD locus is processed. This enables a Bayesian genotype caller to provide precise SNPs, and a robust algorithm to phase those SNPs into long haplotypes, generating RAD loci that are 400–800 bp in length. To prove its recall and precision, we tested the software with simulated data and compared reference‐aligned and de novo analyses of three empirical data sets. Our study shows that the latest version of Stacks is highly accurate and outperforms other software in assembling and genotyping paired‐end de novo data sets.

A GT-seq panel for walleye (Sander vitreus) provides a generalized workflow for efficient development and implementation of amplicon panels in non-model organisms

“…Loci were identified and genotyped in STACKS v.2.2 (Rochette et al, 2019) without using gapped alignments. Raw reads were demultiplexed and barcodes were trimmed in process_radtags (parameter flags: -e SbfI , -c, -q, -filter_illumina, -r, --bestrad).…”

Accuracy of de novo assembly of DNA sequences from double‐digest libraries varies substantially among software

“…We included four assemblers that use graph‐based algorithms in our comparison: Stacks (version 1.46), Stacks2 (version 2.1), ABySS (version 1.3.4) and Velvet (version 1.1) (Catchen et al, ; Rochette et al, ; Simpson et al, ; Zerbino & Birney, ). We evaluated both Stacks and Stacks2 due to significant changes in the software related to how insertion and deletion (indel) variation is treated (changes to Stacks2 since the version 2.1 we used have not included changes to de novo assembly).…”

“…We report the software that was used in those studies and the extent to which papers evaluated and reported different assemblies based on different software settings and parameters. Some software was designed specifically for assembling RAD sequences (e.g., Stacks ; Catchen, Amores, Hohenlohe, Cresko, & Postlethwait, ; Rochette, Rivera‐Colon, & Catchen, ). Other programs were designed for assembly of whole genomes, transcriptomes, or protein sequences, including assembling contiguous sequences (contigs) that are longer than raw reads from the sequencing instrument (e.g., Velvet ; Zerbino & Birney, ).…”

Genotyping by RAD Sequencing Analysis Assessed the Genetic Distinctiveness of Experimental Lines and Narrowed down the Genomic Region Responsible for Leaf Shape in Endive (Cichorium endivia L.)

“…Raw reads obtained through an Ion S5 sequencer (Thermo Fisher Scientific Inc., Waltham, MA, USA) were trimmed according to the enzyme recognition sequence, and after a quality check, all the artifacts and the Ns-containing reads were removed. Variants were called using the Stacks v2.41 software [25], and SNPs were filtered to remove those meeting the following criteria: (1) SNPs with more than 10% missing data, (2) SNPs with a sequence depth ≤ 4, and (3) tri-and tetra-allelic SNPs.…”