Editing NGS Library Preparation (section)

==== Data analysis. ====

Reference sequences for each organism were obtained from GenBank. For Norovirus the sequences were GII.4, and the Norovirus Hu/GII.4/Orange/NSW001P/2008/AU was used as a reference (accession number GQ845367). This was selected as the most common match to the Sanger sequenced 288 bp RT-PCR amplicon used in the diagnostic RT-PCR stage. For HCV the reference sequence used was the subtype 3a strain (accession number AF046866).



Sequences were mapped to the organism-specific reference using Stampy v1.0.14 [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Lunter1 [25]] with no BWA pre-mapping. Bases and single nucleotide variants (SNVs) were called using the SAMtools “mpileup” command with options ‘-E -M0 -Q30 -q30 -o40 -e20 -h100 -m2 -D -S’ and BCFtools [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Li1 [26]]. Sites were filtered to avoid unreliable calls using the following criteria:

#A minimum depth of 5 reads at each position
#A minimum average base quality of 10
#A minimum SNV quality of 25
#At least 75% of reads at the position support the call and the position was called as homozygous



Positions were called as heterozygous based on the SAMtools genotype calls. An evolutionary tree was created using BEAST (Bayesian evolutionary analysis sampling trees) depicting all the full genomic sequences with relatedness. Analysis was performed using BEAST v.1.7.5 combining two random number seed chains (10 million iterations each, saving 1 in 1,000 iterations, with a 1 million iteration burn-in) using: HKY substitution; estimated frequency; strict clock; and constant population size coalescent tree prior. This maximum clade credibility tree was computed using TreeAnnotator v.1.7.5 and plotted with Figtree v.1.4.0. [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066129#pone.0066129-Drummond1 [27]].



The sequences reported in this paper have been deposited in the European Nucleotide Archive Sequence Read Archive under study accession number ERP002219. The Sanger sequences used for validation for Norovirus comparison have been submitted to EMBL under accession numbers HF952119-HF952135. For HCV, the Sanger sequences used for comparison were Genbank accession numbers GQ356201 and KC836883.
[[Category:Protocols]]