A high-throughput Sanger strategy for human mitochondrial genome sequencing

A population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets. We developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences. The described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing.https://doi.org/10.1186/1471-2164-14-88

Evaluation of Modified Yfiler™ Amplification Strategy for Compromised Samples

amplification protocol for the AmpFSTR® Yfiler™ PCR Amplification Kit (Applied Biosystems) and explore the potential of Y-chromosomal short tandem repeat (Y-STR) recovery from severely degraded skeletal remains encountered at the Armed Forces DNA Identification Laboratory. Methods Experiments were performed using two sets of Yfiler™ amplification parameters. One set of parameters reflected the manufacturer’s recommendations. The second set of parameters included twice the recommended Taq concentration and 6 additional cycles. Recovery of authentic alleles and the incidence of drop-in alleles were assessed for 3 data sets: 8 different quantities of pristine DNA, 8 artificially-degraded samples, and 31 non-probative case samples. Results Samples tested with both protocols from all 3 data sets yielded twice as many authentic alleles under the modified parameters than under the standard parameters (62% vs 31%), with only a nominal associated increase in the occurrence of non-authentic alleles (1.36% of all alleles detected). When applied to a range of representative casework samples, the modified protocol leveraged 9 or more reproducible alleles from over half of the specimens tested. Conclusion Reproducible and informative Y-STR profiles can be recovered from a broad range of degraded and inhibited skeletal remains extracts when a commercially available kit is employed under modified amplification parameters