Effects and considerations of multiplexing ForenSeq Kintelligence libraries with a negative control.

The negative template control or negative amplification control has been an essential component of the forensic DNA analysis workflow that helps monitor contamination. As such, the inclusion of a negative control in forensic DNA analysis has been a requirement for all laboratories audited under the FBI's Quality Assurance Standards. As massively parallel sequencing (MPS) becomes more conventional in forensic laboratories, considerations for the inclusion of a negative control in every sequencing run can be evaluated. Although the inclusion of a negative control in library preparation and the first sequencing run has a practical function, there is less utility for its inclusion in all subsequent sequencing runs for that library preparation. Although this is universal to all MPS assays, it is most relevant for an assay that has a low sample multiplexing capacity, such as the ForenSeq Kintelligence Kit (Qiagen/Verogen, Inc.). The ForenSeq Kintelligence Kit is an investigative genetic genealogy (IGG) sequencing-based assay that targets 10,230 forensically relevant single-nucleotide polymorphisms. The manufacturer recommends multiplexing 3 libraries per sequencing run, which includes controls. The purpose of this study was to investigate the effect of the inclusion of a negative control in every Kintelligence sequencing run. We observed that the library generated from a negative amplification control will take 7%-14% of the run output. The loss of sequencing space taken by a negative control decreased the available output for DNA-containing samples, leading in some cases to allele or locus dropout and accompanying higher numbers of sixth to seventh order unknown associations in GEDmatch PRO.

Noninvasive Prenatal Test for ß-Thalassemia and Sickle Cell Disease Using Probe Capture Enrichment and Next-Generation Sequencing of DNA in Maternal Plasma.

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 ß-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 ß-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 ß-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 ß-hemoglobinopathies.