engleski

High-quality sequences of whole mitochondrial genomes from Illumina MiSeq

Analysis of mitochondrial DNA (mtDNA) for forensic applications requires high-quality data. Our aim in this study was to obtain whole mtDNA sequences of sufficient quality in order to produce confident haplogroup determination for future database use. Reference samples of buccal swabs from 23 donors were extracted and purified on EZ1® Advanced XL (Qiagen®). Following PCR amplification of two overlapping fragments (9.1 and 11.2 kbp) for each sample, library preparation was performed with Nextera® XT Sample Prep Kit (Illumina®). Sequencing was run on MiSeq® (Illumina®) instrument, using MiSeq® Reagent kit v2. Analysis was performed with on-instrument software MiSeq® Reporter v2.5.1 and Sequencing Analysis Viewer v.1.8.46, and subsequently with applications from BaseSpace® Sequence Hub (Illumina®). Haplogroups were determined by HaploGrep2 tool. Results exhibited 92.9% of reads with Q-score above 30 and average coverage depth of 9, 928 reads. Composition of haplogroups showed predominance of group H (44%), which is concordant with European haplogroup frequencies. Also, high-level heteroplasmies were detected in 5 samples. In conclusion, we obtained high-quality whole mtDNA sequences, which enabled confident variant calling and haplogroup determination. Further sequencing of larger Croatian population sample is under way, aimed at founding national database of whole mtDNA sequences.

NGS ; Whole mtDNA sequences ; Illumina MiSeq ; Data analysis ; Mitochondrial haplogroups

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