UGbS-Flex, a novel bioinformatics pipeline for imputation-free SNP discovery in polyploids without a reference genome: finger millet as a case study

Abstract

BackgroundResearch on orphan crops is often hindered by a lack of genomic resources. With the advent of affordable sequencing technologies, genotyping an entire genome or, for large-genome species, a representative fraction of the genome has become feasible for any crop. Nevertheless, most genotyping-by-sequencing (GBS) methods are geared towards obtaining large numbers of markers at low sequence depth, which excludes their application in heterozygous individuals. Furthermore, bioinformatics pipelines often lack the flexibility to deal with paired-end reads or to be applied in polyploid species.ResultsUGbS-Flex combines publicly available software with in-house python and perl scripts to efficiently call SNPs from genotyping-by-sequencing reads irrespective of the species’ ploidy level, breeding system and availability of a reference genome. Noteworthy features of the UGbS-Flex pipeline are an ability to use paired-end reads as input, an effective approach to cluster reads across samples with enhanced outputs, and maximization of SNP calling. We demonstrate use of the pipeline for the identification of several thousand high-confidence SNPs with high representation across samples in an F3-derived F2 population in the allotetraploid finger millet. Robust high-density genetic maps were constructed using the time-tested mapping program MAPMAKER which we upgraded to run efficiently and in a semi-automated manner in a Windows Command Prompt Environment. We exploited comparative GBS with one of the diploid ancestors of finger millet to assign linkage groups to subgenomes and demonstrate the presence of chromosomal rearrangements.ConclusionsThe paper combines GBS protocol modifications, a novel flexible GBS analysis pipeline, UGbS-Flex, recommendations to maximize SNP identification, updated genetic mapping software, and the first high-density maps of finger millet. The modules used in the UGbS-Flex pipeline and for genetic mapping were applied to finger millet, an allotetraploid selfing species without a reference genome, as a case study. The UGbS-Flex modules, which can be run independently, are easily transferable to species with other breeding systems or ploidy levels.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1316-3) contains supplementary material, which is available to authorized users.

Genetic Diversity and Population Structure of Rhododendron canescens, a Native Azalea for Urban Landscaping

“…DNA samples (250 ng) were digested with Msp1 and Pst1 at 37°C for 2 h in a 96-well plate. Barcoded PstI adapters and MspI Y-adapters were ligated to the digested DNA fragments, as described in Qi et al, (2018). Small DNA fragments (<400 bp) were eliminated using a Mag-Bind RxnPure Plus kit (Omega Bio-Tek).…”

Population genetic structure and gene flow of rare and endangered Tetraena mongolica Maxim revealed by reduced representation sequencing

“…we adopted modified GBS methods (SuperGBS, following Qi et al [43]) to construct libraries, briefly, extracted DNA from each individual was digested with both PstI-HF and MspI (New England Biolabs, NEB) for 2 h at 37 °C and subsequent 2 h incubation at 75 °C. The barcoded adapters and common adapter were respectively ligated on the PstI cut site and the MspI cut site of all samples by T4 DNA ligase (NEB), ligation was run at 22 °C for 2 h. Fragments smaller than 300 bp were removed using recovery system of improved magnetic bead.…”

“…Because the public databases does not contain full genome information for TM, and even there is also no the genome data of relative species of zygophyllaceae family to reference, under these circumstances, de novo generation of a GBS reference was constructed following [43]. Clean Reads were aligned against the GBS reference genome using bowtie2 (v2.3.4.1) software (main parametermaxins 300 -no-discordant -no-mixed) [46], then genotyping was performed applying Unified program in the GATK (v3.8-1) software (Main parameters -dcov 1000-GLM BOTH) [47] to predict SNP&INDEL sites in samples, the predicted results are screened using the SelectVariants program of the GATK software (Key parameters: -restricelesto biallelic-select "QD > 10.0"), in order to reduce the error rate of detected SNP&INDEL, we used the software vcftools (v0.1.13) (main parameter -maf 0.01 -minDP 4 -max-missing 0.8) to filter the obtained SNP typing results.…”

Population genetic structure and gene flow of rare and endangered Tetraena mongolica Maxim revealed by reduced representation sequencing

“…Because the public databases does not contain full genome information for TM, and even there is also no the genome data of relative species of zygophyllaceae family to reference. Under these circumstances, de novo generation of a GBS reference was constructed following [43]. Clean Reads were aligned against the GBS reference genome using bowtie2 (v2.3.4.1) software (main parameter -maxins 300no-discordant -no-mixed) [46], and then genotyping was performed applying Uni ed program in the GATK (v3.8-1) software (Main parameters -dcov 1000-GLM BOTH) [47] to predict SNP&INDEL sites in samples.…”