Intra‐individual polymorphism in chloroplasts from NGS data: where does it come from and how to handle it?

Abstract

Next-generation sequencing allows access to a large quantity of genomic data. In plants, several studies used whole chloroplast genome sequences for inferring phylogeography or phylogeny. Even though the chloroplast is a haploid organelle, NGS plastome data identified a nonnegligible number of intra-individual polymorphic SNPs. Such observations could have several causes such as sequencing errors, the presence of heteroplasmy or transfer of chloroplast sequences in the nuclear and mitochondrial genomes. The occurrence of allelic diversity has practical important impacts on the identification of diversity, the analysis of the chloroplast data and beyond that, significant evolutionary questions. In this study, we show that the observed intra-individual polymorphism of chloroplast sequence data is probably the result of plastid DNA transferred into the mitochondrial and/or the nuclear genomes. We further assess nine different bioinformatics pipelines' error rates for SNP and genotypes calling using SNPs identified in Sanger sequencing. Specific pipelines are adequate to deal with this issue, optimizing both specificity and sensitivity. Our results will allow a proper use of whole chloroplast NGS sequence and will allow a better handling of NGS chloroplast sequence diversity.

Pre‐Pleistocene origin of phylogeographical breaks in African rain forest trees: New insights from Greenwayodendron (Annonaceae) phylogenomics

“…Samtools 1.1 (Li & Durbin, ) was used to generate a mpileup file with option ‐B, following Scarcelli et al. (). We used Varscan 2.3.7 (Koboldt et al., ) to call SNPs (options min‐var‐freq 0.01, min‐avg‐qual 30, min coverage 5) before converting the resulting vcf file into a fasta multi‐alignment file and removing heterozygous sites.…”

“…The mapping of plastid DNA-enriched libraries on this reference plastome was performed using BWA MEM 0.7.12 (Li, 2013;Li & Durbin, 2009) with -M and -B 4 options. SAMTOOLS 1.1 (Li & Durbin, 2009) was used to generate a mpileup file with option -B, following Scarcelli et al (2016). We used VARSCAN 2.3.7 (Koboldt et al, 2012) to call SNPs (options min-var-freq 0.01, min-avg-qual 30, min coverage 5) before converting the resulting vcf file into a fasta multi-alignment file and removing heterozygous sites.…”

Human management and hybridization shape treegourd fruits in the Brazilian Amazon Basin

“…Fragment size and allele identification were determined using GeneMapper (Applied Biosystems). We also obtained SNPs observed in the whole chloroplast using previously described approaches (Moreira et al, 2016;Scarcelli et al, 2016).…”

Guinea yam (Dioscorea spp., Dioscoreaceae) wild relatives identified using whole plastome phylogenetic analyses

“…Reconstruction of whole plastomes. -Raw reads were processed and filtered according to Scarcelli & al. (2016) using Demultadapt (https://github.com/Maillol/demultadapt) for demultiplexing, Cutadapt v.1.2.1 (Martin, 2011) for trimming of adapters and low quality bases and a free Python script (https://github.com/SouthGreenPlatform/arcad-hts/blob/master/ scripts/arcad_hts_2_Filter_Fastq_On_Mean_Quality.pl) for read quality filtering.…”