DOI: 10.5061/dryad.t80gj
title: Data from: Whole genome sequence accuracy is improved by replication in a population of mutagenized sorghum.

description: The accurate detection of induced mutations is critical for both forward
 and reverse genetics studies. Experimental chemical mutagenesis induces
 relatively few single base changes per individual. In a complex eukaryotic
 genome, false positive detection of mutations can occur at or above this
 mutagenesis rate. We demonstrate here, using a population of ethyl
 methanesulfonate (EMS) treated Sorghum bicolor BTx623 individuals, that
 using replication to detect false positive induced variants in
 next-generation sequencing data permits higher throughput variant
 detection with greater accuracy. We used a lower sequence coverage depth
 (average of 7X) from 586 independently mutagenized individuals and
 detected 5,399,493 homozygous SNPs. Of these, 76% originated from only
 57,872 genomic positions prone to false positive variant calling. These
 positions are characterized by high copy number paralogs where the
 error-prone SNP positions are at copies containing a variant at the SNP
 position. The ability of short stretches of homology to generate these
 error prone positions suggests that incompletely assembled or poorly
 mapped repeated sequences are one driver of these error prone positions..
 Removal of these false positives left 1,275,872 homozygous and 477,531
 heterozygous EMS-induced SNPs which, congruent with the mutagenic
 mechanism of EMS, were greater than 98% G:C to A:T transitions. Through
 this analysis we generated a database of sequence indexed mutants of
 Sorghum. This collection contains 4,035 high impact homozygous mutations
 in 3,637 genes and 56,514 homozygous missense mutations in 23,227 genes.
 Each line contains, on average, 2,177 annotated homozygous SNPs per
 genome, including seven likely gene knockouts and 96 missense mutations.
 The number of mutations in a transcript was linearly correlated with the
 transcript length and also the G+C count, but not with the GC/AT ratio.
 Analysis of the detected mutagenized positions identified CG-rich patches,
 and flanking sequences strongly influenced EMS-induced mutation rates. Our
 method for detecting false-positive induced mutations is generally
 applicable to any organism, is independent of the choice of in silico
 variant-calling algorithm, and is most valuable when the true mutation
 rate is likely to be low, such as in laboratory induced mutations or
 somatic mutation detection in medicine.
description: Supplemental File
 S1Standard filtered SNPs
 detected in each of the 586 sorghum individuals.Supplemental_File-S1.zipSupplemental File S2Probable error-prone SNP genomic positions in the sorghum reference genome (version 2.1).Supplemental_File-S2.zipSupplemental File S3Non-replicate and likely EMS-induced homozygous SNPs in each of the 586 sorghum individuals.Supplemental_File-S3.zipSupplemental File S4Non-replicate, and likely EMS-induced heterozygous SNPs in each of the 586 sorghum individuals.Supplemental_File-S4.zipSupplemental File S5Counts of homozygous and heterozygous SNPs in all the 586 sequenced lines.Supplemental_File-S5.zipSupplemental File S6Replicate and likely false-negative EMS-induced homozygous G:C to A:T SNPs in each of the 586 sorghum individuals.Supplemental_File-S6.zipSupplemental File S7Replicate and likely false-negative EMS-induced heterozygous G:C to A:T SNPs in each of the 586 sorghum individuals.Supplemental_File-S7.zipSupplemental File S8SnpEff functional classification of the homozygous EMS-induced SNPs in all the 586 sorghum individuals.Supplemental_File-S8.zipSupplemental File S9Function description for the genes containing the SnpEff-annotated homozygous EMS-induced SNPs in all the 586 sorghum individuals.Supplemental_File-S9.zipSupplemental File S10List of SnpEff-annotated EMS-induced SNPs predicted to trigger silent stop codon lost substitutions in genes of the 586 sorghum individuals.Supplemental_File-S10.zipSupplemental File S11SnpEff functional classification of the heterozygous EMS-induced SNPs in all the 586 sorghum individuals.Supplemental_File-S11.zipSupplemental File S12Function description for the genes containing the SnpEff-annotated heterozygous EMS-induced SNPs in all the 586 sorghum individuals.Supplemental_File-S12.zipSupplemental File S13Function description for the genes containing the SnpEff-annotated tentative false-negative homozygous EMS-induced SNPs in all the 586 sorghum individuals.Supplemental_File-S13.zipSupplemental File S14Function description for the genes containing the SnpEff-annotated tentative false-negative heterozygous EMS-induced SNPs in all the 586 sorghum individuals.Supplemental_File-S14.zipSupplemental File S15SnpEff functional classification of the likely EMS-induced indels in all the 586 sorghum individuals.Supplemental_File-S15.zipSupplemental File S16Function description for the genes containing the SnpEff-annotated likely EMS-induced indels in all the 586 sorghum individuals.Supplemental_File-S16.zipSupplemental File S17SIFT prediction results for the missense-annotated homozygous EMS-induced SNPs in all the 586 sequenced individuals.Supplemental_File-S17.zipSupplemental File S18SIFT prediction results for the missense-annotated heterozygous EMS-induced SNPs in all the 586 sequenced individuals.Supplemental_File-S18.zipSupplemental File S19Summary statistics for the sequencing, mapping, variants prediction, filtering, annotation and classification for the detected EMS-induced variants in all 586 sequenced individuals.Supplemental_File-S19.zipSupplemental File S20Detailed classification, gene function description and annotation of the medium or high impact homozygous EMS-induced SNPs.Supplemental_File-S20.zipSupplemental File S21Detailed classification, gene function description and annotation of the medium or high impact heterozygous EMS-induced SNPs.Supplemental_File-S21.zipSupplemental File S22Detailed classification, gene function description and annotation of the medium or high impact EMS-induced indels.Supplemental_File-S22.zipSupplemental File S23The detailed attributes of gene transcripts, including number of SNPs, GC-content, sequence length, mononucleotide and dinucleotide counts, which were used in the linear correlation analysis.Supplemental_File-S23.zipSupplemental File S24The subset of BLAST ungapped global alignments for the 51nt sequence contexts of the error-prone SNP positions, randomly selected EMS-induced SNP positions and randomly selected genome positions.Supplemental_File-S24.zipSupplemental File S25Sample scripts for the variants detection and annotation pipeline.Supplemental_File-S25.zip