DOI: 10.5061/dryad.cd711
title: Data from: Impact of enrichment conditions on cross-species capture of fresh and degraded DNA

description: By combining high-throughput sequencing with target-enrichment
 (“hybridization capture”), researchers are able to obtain molecular data
 from genomic regions of interest for projects that are otherwise
 constrained by sample quality (e.g. degraded and contamination-rich
 samples) or a lack of a priori sequence information (e.g. studies on
 non-model species). Despite the use of hybridization capture in various
 fields of research for many years, the impact of enrichment conditions on
 capture success are not yet thoroughly understood. We evaluated the impact
 of a key parameter – hybridization temperature – on the capture success of
 mitochondrial genomes across the carnivoran family Felidae. Capture was
 carried out for a range of samples types (fresh, archival, ancient) with
 varying levels of sequence divergence between bait and target (i.e. across
 a range of species) using pools of individually indexed libraries on
 Agilent SureSelect arrays. Our results suggest that hybridization capture
 protocols require specific optimization for the sample type that is being
 investigated. Hybridization temperature affected the proportion of
 on-target sequences following capture: for degraded samples, we obtained
 the best results with a hybridization temperature of 65 °C, while a
 touchdown approach (65 °C down to 50 °C) yielded the best results for
 fresh samples. Evaluation of capture performance at a regional scale
 (sliding window approach) revealed no significant improvement in the
 recovery of DNA fragments with high sequence divergence from the bait at
 any of the tested hybridization temperatures, suggesting that
 hybridization temperature may not be the critical parameter for enrichment
 of divergent fragments.
description: Ancestral mitogenome
 reconstructionAs interspecific
 comparisons featured heavily in our analyses, it was important to choose a
 reference sequence that would limit the introduction of a bias during the
 mapping of sequences (“mapping bias”). Mapping algorithms become less
 effective when the reference is very dissimilar (Prüfer et al. 2010;
 Schubert et al. 2012) impacting the number of sequences identified as
 ‘on-target’. As some of the species in this study currently do not have a
 reference sequence available from GenBank (Table 1), we reconstructed an
 ancestral mitogenome of all Felidae (Appendix S1; Supporting Table S2) to
 be used as reference. To reconstruct the ancestral sequence, the most
 appropriate model of substitution for the alignment of the felid
 mitogenomes was estimated using jModeltest 2.1.4 under the Bayesian
 Information Criterion (Posada 2008), which was then implemented in PhyML
 (Guindon et al. 2010) to infer the topology of the phylogeny (as shown in
 Fig. 1A). Reconstruction of the ancestral sequence was performed using the
 webserver of Ancestors v1.1 (Diallo et al. 2007, 2010). For additional
 information, please refer to the manuscript and its
 supplements.felids_ancestral_29.11.2013.fastaPerl script 1Perl script written by Kanchon Dasmahapatra (University of York; kanchon.dasmahapatra@york.ac.uk). This script uses a variant file (VCF) generated by GATK to call the individual bases per position for a haploid genome. After this script, run "convert_callsfile_to_one_line_fasta.pl" script to covert the base calls to a fasta file.filter_multiple_sample_gatk1.5_vcf_mtDNA_indels.plPerl script 2Perl script written by Kanchon Dasmahapatra (University of York; kanchon.dasmahapatra@york.ac.uk). This script uses the output from "filter_multiple_sample_gatk1.5_vcf_mtDNA_indels.pl" and writes the base calls to a fasta file.convert_callsfile_to_one_line_fasta.plTree filesTree files for the phylogenies displayed in Supporting Figure 5. Phylogenies were generated using PhyML.Tree-files.zipIncomplete mitogenomes for Neofelis Nebulosa and Leptailurus servalThis zip file contains two fasta files with the incomplete mitogenomes for Neofelis Nebulosa ("NNE_Fell") and Leptailurus serval ("LSE_Togo"). These mitogenomes were too low quality for upload on GenBank: LSE_Togo is 44.5% complete and NNE_Fell is 51.0% complete. The length of stretches of N's are estimated based on a closely related individual. For more information regarding these sequences, feel free to contact the authors.LSE-NNE_incomplete-mitogenomes.zip