title: Genome sequencing of P. andina isolate PaX: Sample PaX- Aligned reads only
design: Modern DNA extracts were sheared to a mean fragment length of 200-400bp using a Diagenode Bioruptor XL and then purified using a MinElute PCR purification kit. Sheared DNA extracts were built into Illumina sequencing libraries using a NEBNext DNA Library Preparation for Roche/454 kit and following manufacturers’ protocols except that MinElute PCR purification columns were used, rather than a streptavidin beads protocol. Blunt-end adapters P5 and P7 were prepared according to Meyer and Kircher (2010). Initial DNA amplification was carried out through 12 cycles of PCR in 50-µL reactions containing Phusion Hi-Fidelity PCR Master Mix, 0.2 µMol primer inPE1.0, 0.2 µMol Illumina PCR reverse primer with unique index and 5µL library as DNA template. PCR products were purified using MinElute purification columns and size-selected using a Caliper LabChip XT.   Post-extraction and pre-PCR manipulations of historical DNA were carried out in dedicated ancient DNA laboratories at the Centre for GeoGenetics, University of Copenhagen. Historical DNA extracts were built into libraries as described above, except that shearing was not performed and adapter concentration was reduced from manufacturers’ specifications during adapter ligation. Unique index sequences were added through PCR amplification of libraries. Initial DNA amplification was carried out through 10 cycles of PCR in two parallel 50-µL reactions containing 5 units AmpliTaq Gold polymerase, 5 µL 1X PCR Gold buffer, 4 mMol MgCl2, 50 µg BSA, 1 mMol each dNTP, 12.5 µMol inPE1.0 forward primer, 10 nMol inPE2.0 reverse primer, 12.5 nMol unique index primer, and 15 µL DNA library template. Amplified PCR products were purified on MinElute purification columns and eluted in 23 µL EB buffer. 5 µL of this purified product was used as template in another round of PCR that was otherwise the same, except that primer inPE2.0 was not included. These amplified PCR products were purified on MinElute purification columns and size-selected using a Caliper LabChip XT.  The command-line pipeline PALEOMIX was used to perform systematic adapter trimming, read mapping, and mapped filtering (Schubert et al. 2014). Low-quality bases and residual adapter sequences (inferred from library building methods or obtained directly from authors) were trimmed from the 3’ ends of DNA reads with the software AdapterRemoval v1.5 (Lindgreen 2012) using a mismatch rate of 0.333 (command-line option --mm 3). Paired reads overlapping by at least 11bp were collapsed into a single read with a re-calibrated base quality. Reads shorter than 25bp after adapter sequence removal were discarded.  Release ASM14294v1 of the draft reference nuclear genome assembly and annotation of P. infestans strain T30-4 (Haas et al. 2009) were obtained from the EnsemblProtists Phytophthora infestans assembly and gene annotation database (protists.ensembl.org/Phytophthora_infestans/). Mapping to the reference genome was performed with Burrows-Wheeler Aligner (BWA) v0.5.10 (Li and Durbin 2009) with seeding deactivated and otherwise default settings. PCR duplicates were removed with the MarkDuplicates function within the software Picard v1.82. Collapsed reads were filtered using a script included with PALEOMIX. Reads with mapping quality (MAPQ) scores < 25 were removed from further analysis. Read alignments to the reference genome were optimized with the RealignerTargetCreator and IndelRealigner tools included in the software Genome Analysis Toolkit (GATK) v1.3 (McKenna et al. 2010). This pipeline also utilizes MapDamage2 (Jónsson et al. 2013) to recalibrate base qualities of aligned sequence reads in order to remove the residual chemical damage pattern characteristic to ancient DNA.