Introduction | Workflow

Workflow

The mtDNA Variant Processor App supports both mtDNA D-Loop, custom regions, and whole genome samples. See Analysis Methods.

 

Nextera Adapter Trimming

▶ Nextera adapters are removed from the forward and reverse reads.
▶ Trimming is repeated until there are no more than 3 adapter bases on the end of the read.
▶ Reads are discarded when they are excessively trimmed or have short amplicons.

Alignment

▶ BWA-MEM performs the alignment.
▶ Circular alignment is handled across the origin by identifying the true start and end of reads.
▶ Realigns regions with indels to improve alignment and shifts indels to a 3' alignment.

Account for Primers

▶ Manifest identifies the primers and amplicons.
▶ Removes primer contributions from the reads for accurate variant calling.

Variant Calling

▶ Identifies nucleotides that are greater than the interpretation threshold for the position.
▶ Generates percentage of reads for a nucleotide that exceeds the analysis threshold (at).
▶ Generates a percentage of reads for a nucleotide that exceeds the interpretation threshold (it).
▶ Generates the interpretation threshold indicator for positions that have a nucleotide between the analysis threshold and the interpretation threshold.
▶ Reports base call codes. See Base Call Codes.

Filtering and Scoring

▶ Removes reads that do not meet the minimum quality score for a base call.
▶ Calculates a score from the minimum quality score for base call, alignment score, and presence of low abundance reads at the position.

Output Files

▶ VCF files and BAM files.
▶ Use the mtDNA Variant Analyzer App to view the analysis results.