Mitigating the effects of reference sequence bias in single-multiplex massively parallel sequencing of the mitochondrial DNA control region.

Sequence analysis of the mitochondrial DNA (mtDNA) control region can provide forensically useful information, particularly in challenging samples where autosomal DNA profiling fails. Sub-division of the 1122-bp region into shorter PCR fragments improves data recovery, and such fragments can be analysed together via massively parallel sequencing (MPS). Here, we generate mtDNA data using the prototype PowerSeq™ Auto/Mito/Y System (Promega) MPS assay, in which a single PCR reaction amplifies ten overlapping amplicons of the control region, in a set of 101 highly diverse samples representing most major clades of the mtDNA phylogeny. The overlapping multiplex design leads to non-uniform coverage in the regions of overlap, where it is further increased by short amplicons generated alongside the intended products. Primer sequences in targeted amplification libraries are a potential source of reference sequence bias and thus should be removed, but the proprietary nature of the primers in commercial kits necessitates an alternative approach that minimises data loss: here, we introduce the bioinformatic selection of sequencing reads spanning putative primer sites (Overarching Read Enrichment Option, OREO). While OREO performs well in mitigating the effects of primer sequences at the ends of sequence reads, we still find evidence of the internalisation of primer-derived sequences by overlap extension, which may compromise the ability to call variants or to measure heteroplasmy in primer-binding regions. The commercially available PowerSeq™ CRM Nested System design prevents primer internalisation, as shown in a reanalysis of a subset of 57 samples that contain possible heteroplasmies. In combination with OREO, the CRM Nested kit mitigates reference sequence bias, allowing heteroplasmic variants to be estimated down to a 5% threshold. Provided appropriate steps are taken in data processing, single-reaction multiplex assays represent robust tools to analyse mtDNA control region variation. The OREO approach will allow users to bypass the effects of unknown primer sequences in any single-reaction tiled multiplex and eliminate primer-derived bias in overlapping amplicon sequencing studies, in both forensic and non-forensic settings

A phylogenetic framework facilitates Y-STR variant discovery and classification via massively parallel sequencing

Short-tandem repeats on the male-specific region of the Y chromosome (Y-STRs) are permanently linked as haplotypes, and therefore Y-STR sequence diversity can be considered within the robust framework of a phylogeny of haplogroups defined by single-nucleotide polymorphisms (SNPs). Here we use massively parallel sequencing (MPS) to analyse the 23 Y-STRs in Promega’s prototype PowerSeqÔ Auto/Mito/Y System kit (containing the markers of the PowerPlex® Y23 [PPY23] System) in a set of 100 diverse Y chromosomes whose phylogenetic relationships are known from previous megabase-scale resequencing. Including allele duplications and alleles resulting from likely somatic mutation, we characterised 2311 alleles, demonstrating 99.83% concordance with capillary electrophoresis (CE) data on the same sample set. The set contains 267 distinct sequence-based alleles (an increase of 58% compared to the 169 detectable by CE), including 60 novel Y-STR variants phased with their flanking sequences which have not been reported previously to our knowledge. Variation includes 46 distinct alleles containing non-reference variants of SNPs/indels in both repeat and flanking regions, and 145 distinct alleles containing repeat pattern variants (RPV). For DYS385a,b, DYS481 and DYS390 we observed repeat count variation in short flanking segments previously considered invariable, and suggest new MPS-based structural designations based on these. We considered the observed variation in the context of the Y phylogeny: several specific haplogroup associations were observed for SNPs and indels, reflecting the low mutation rates of such variant types; however, RPVs showed less phylogenetic coherence and more recurrence, reflecting their relatively high mutation rates. In conclusion, our study reveals considerable additional diversity at the Y-STRs of the PPY23 set via MPS analysis, demonstrates high concordance with CE data, facilitates nomenclature standardisation, and places Y-STR sequence variants in their phylogenetic context