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Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with the advent of massively parallel sequencing (MPS) technology. Knowledge of the particular workflow, equipment, and reagents used, along with extensive usage of negative controls to monitor all preparation steps constitute the prerequisites for confident reporting of results. In this study, we performed an assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis of several types of negative controls, as well as mtDNA positive controls, we established thresholds for data analysis and interpretation, consisting of several components: minimum read depth (220 reads), minimum quality score (41), percentage of minor allele sufficient for analysis (3.0%), percentage of minor allele sufficient for interpretation (6.0%), and percentage of major allele sufficient for homoplasmic variant call (97.0%). Based on these criteria, we defined internal guidelines for analysis and interpretation of mtDNA results obtained by MPS. Our study shows that the whole mtDNA assay on MiSeq FGx™ produces repeatable and reproducible results, independent of the analyst, which are also concordant with Sanger-type sequencing results for mtDNA control region, as well as with MPS results produced by NextSeq®. Overall, established thresholds and interpretation guidelines were successfully applied for the sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on the definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, e.g. targeting good-quality samples in forensic genetics and molecular diagnostics. [ABSTRACT FROM AUTHOR]

Rudstam, L. G., Parker-Stetter, S. L., Sullivan, P. J., and Warner, D. M. 2009. Towards a standard operating procedure for fishery acoustic surveys in the Laurentian Great Lakes, North America. – ICES Journal of Marine Science, 66: 1391–1397.Acoustic surveys are conducted annually in all five of the Laurentian Great Lakes and Lake Champlain to assess forage-fish abundance. The main target species are rainbow smelt (Osmerus mordax), alewife (Alosa pseudoharengus), and several coregonine species (Coregonus spp.). The Great Lakes Fishery Commission sponsored an Acoustic Study Group from 2002 to 2006 to discuss common problems and suggest standardized methods across these lakes. The study group produced a set of recommendations, available as a Great Lakes Fishery Commission Special Publication and on the web, that use in situ target strength (TS) to scale volume backscattering. Here, we review these recommendations with special attention to four often-overlooked topics of interest to all acoustic users, namely issues associated with first, the choice of thresholds for both TS and volume-backscattering strength, second, different settings for single-echo detection algorithms for measures of in situ TS, third, those taking account of measuring in situ TS in dense fish concentrations, and finally, detection limits. [ABSTRACT FROM PUBLISHER]