Quality Human Mitogenome Sequence Data Amenable for Use as Population Reference Data Obtained from Manual Processing Using the Collibri™ ES DNA Library Prep Kit

Mitochondrial DNA (mtDNA) sequencing is a crucial tool in forensic investigations. Although Sanger-type sequencing is the gold standard method, it is labor-intensive, costly, and has low throughput, limiting its adoption for routine use in forensic laboratories. Next-generation sequencing coupled with automation has made cost-effective high-throughput sequencing a reality. Earlier methods for the generation of quality mitogenome population-based reference databases for forensic purposes have been centered on automation. While this may be ideal, not all laboratories have automation capabilities. In this study, we report on high-quality mitogenome reference sequence data generated from manual processing using the Collibri™ ES DNA Kit. Long-range PCR was used for target enrichment, the CLC Genomics Workbench (v.22) and AQME (AFDIL-QIAGEN mtDNA Expert) were used for data analysis. The Collibri™ ES DNA Library Prep Kit produced sufficient data (≈12G) when 96 samples were sequenced concurrently. The average depth of coverage was 4403×. Even and high depth of coverage was observed across the entire target region ensuring low-level variant calling at 5% variant frequency. Contamination was not detected and only two samples failed to yield sequence data. Manual library processing using the Collibri™ ES DNA Library Prep Kit on the Illumina Miseq™ produced high-quality data. The Collibri kit reagent components are colored, enhancing the monitoring of workflow progress. The high-quality data coupled with visual quality control makes the kit ideal for manual processing. This protocol is therefore a suitable alternative for generating population-based mitogenome data in settings that have not implemented automation.