The application of next generation sequencing (NGS) for the analysis of mitochondrial (mt) DNA, short tandem repeats (STRs), and single nucleotide polymorphism (SNPs) has demonstrated great promise for challenging forensic specimens, such as degraded, limited, and mixed samples. Target enrichment using probe capture rather than PCR amplification offers advantages for analysis of degraded DNA since two intact PCR primer sites in the template DNA molecule are not required. Furthermore, NGS software programs can help remove PCR duplicates to determine initial template copy numbers of a shotgun library. Moreover, the same shotgun library prepared from a limited DNA source can be enriched for mtDNA as well as nuclear markers by hybrid capture with the relevant probe panels. Here, we demonstrate the use of this strategy in the analysis of limited and mock degraded samples using our custom probe capture panels for massively parallel sequencing of the whole mtgenome and 426 SNP markers. We also applied the mtgenome capture panel in a mixed sample and analyzed using both phylogenetic and variant frequency based bioinformatics tools to resolve the minor and major contributors. Finally, the results obtained on individual telogen hairs demonstrate the potential of probe capture NGS analysis for both mtDNA and nuclear SNPs for challenging forensic specimens.

The authors wish to make the following change to their paper [1][...]

Background Noninvasive prenatal testing (NIPT) of chromosomal aneuploidies based on next-generation sequencing (NGS) analysis of fetal DNA in maternal plasma is well established, but testing for autosomal recessive disorders remains challenging. NGS libraries prepared by probe capture facilitate the analysis of the short DNA fragments plasma. This system has been applied to the β-hemoglobinopathies to reduce the risk to the fetus. Method Our probe panel captures >4 kb of the HBB region and 435 single-nucleotide polymorphisms (SNPs) used to estimate fetal fraction. Contrived mixtures of DNA samples, plasma, and whole blood samples from 7 pregnant women with β-thalassemia or sickle cell anemia mutations and samples from the father, sibling, and baby or chorionic villus were analyzed. The fetal genotypes, including point mutations and deletions, were inferred by comparing the observed and expected plasma sequence read ratios, based on fetal fraction, at the mutation site and linked SNPs. Accuracy was increased by removing PCR duplicates and by in silico size selection of plasma sequence reads. A probability was assigned to each of the potential fetal genotypes using a statistical model for the experimental variation, and thresholds were established for assigning clinical status. Results Using in silico size selection of plasma sequence files, the predicted clinical fetal genotype assignments were correct in 9 of 10 plasma libraries with maternal point mutations, with 1 inconclusive result. For 2 additional plasmas with deletions, the most probable fetal genotype was correct. The β-globin haplotype determined from linked SNPs, when available, was used to infer the fetal genotype at the mutation site. Conclusion This probe capture NGS assay demonstrates the potential of NIPT for β-hemoglobinopathies.

No abstract

No abstract