Raw code to get polymorphisms from samples analysed by using the PowerSeq® CRM Nested System kit (Promega Corporation)

A group of tools must be pre-installed. For each tool, version used by our group is shown in brackets, but another version might work as well:

• FastQC (v0.11.9)
• fastp (v0.23.2)
• BWA (v0.7.17)
• SAMtools (v1.16.1)
• QualiMap (v2.2.2a)
• PMDtools (v0.60)
• freebayes (v1.3.6)
• vcflib (v1.0.3)
• vcftools (v0.1.16)

• Sequencing quality control (FastQC)
• Duplicates removal (fastp)
• Adapter removal (fastp)
• Primers removal (fastp)
• Merge of overlapping Paired End reads (fastp)
• Reads mapping to reference (bwa aln, bwa sampe, samtools)
• Post-mapping processing (samtools)
• BAM quality control (QualiMap)
• Selection of reads with Post-Mortem Damage (PMDtools)
• Obtention of polimorphisms (freebayes + vcfallelicprimitives)

If you use DanielRCA/MTDNA-CR for your analysis, please cite the MTDNA-CR paper as follows:

Vinueza-Espinosa, DC, Cuesta-Aguirre, DR, Malgosa, A, Santos, C. Mitochondrial DNA control region typing from highly degraded skeletal remains by single-multiplex next-generation sequencing. Electrophoresis. 2023; 44: 1423–1434. https://doi.org/10.1002/elps.202200052

References of the tools used:

• FastQC S. Andrews (2010), FASTQC. A quality control tool for high throughput sequence data, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
• fastp S. Chen, Y. Zhou, Y. Chen, J. Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, 1 September 2018, Pages i884–i890, https://doi.org/10.1093/bioinformatics/bty560
• BWA H. Li, R. Durbin, Fast and accurate short read alignment with Burrows-Wheeler transform, Bioinformatics. 25 (2009) 1754–1760. https://doi.org/10.1093/bioinformatics/btp324
• SAMtools P. Danecek, JK. Bonfield, J. Liddle, J. Marshall, V. Ohan, MO. Pollard, A. Whitwham, T. Keane, SA. McCarthy, RM. Davies, H. Li (2021), Twelve years of SAMtools and BCFtools. GigaScience, Volume 10, Issue 2, February 2021, giab008, https://doi.org/10.1093/gigascience/giab008
• QualiMap K. Okonechnikov, A. Conesa, F. García-Alcalde (2016), Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics, Volume 32, Issue 2, 15 January 2016, Pages 292–294, https://doi.org/10.1093/bioinformatics/btv566
• PMDtools P. Skoglund, BH. Northoff, MV. Shunkov, A. Derevianko, S. Pääbo, J. Krause, M. Jakobsson (2014), Separating ancient DNA from modern contamination in a Siberian Neandertal. Proceedings of the National Academy of Sciences USA https://doi.org/10.1073/pnas.1318934111
• freebayes E. Garrison, G. Marth (2012), Haplotype-based variant detection from short-read sequencing. arXiv preprint arXiv:1207.3907 [q-bio.GN]
• vcflib E. Garrison, ZN. Kronenberg, ET. Dawson, BS Pedersen, P Prins (2022), A spectrum of free software tools for processing the VCF variant call format: vcflib, bio-vcf, cyvcf2, hts-nim and slivar. PLoS Comput Biol 18(5): e1009123, https://doi.org/10.1371/journal.pcbi.1009123