Gs reference mapper

Quality control for sequencing data was performed, sequences with a Phred quality score of <20 were eliminated, and primer sequences were trimmed. The Roche GS Reference Mapper (ver. 2.9) was used to identify HPV genotypes by comparing fragment sequences with the L1 region of alpha and beta papillomavirus reference sequences, downloaded from Papillomavirus Episteme (PaVE) [48 ]. Specific reads for each diagnosis group were identified in accordance with their associated MID barcode.

Raw pyrosequencing data was processed by using the default on-rig procedures from 454/Roche. Filter-passing reads were used in the subsequent analyses for each of the pyrosequencing libraries. Sequence reads were de-multiplexed and processed to generate operational taxonomic units (OTU) with the microbial Profiling Using Metagenomic Assembly (mPUMA) pipeline [19 (link)] using Trinity for OTU assembly. Processing of sequence reads by mPUMA includes identification and removal of amplification primer sequences and identification of putative chimeras using the C3 chimera checker. Watered-BLAST [20 (link)] comparison to the cpnDB_nr reference database (version 20130321, downloaded from http://www.cpndb.ca) [21 (link)] was used to identify each OTU. Sequences identified as non-target were reference mapped (GS Reference Mapper, Roche, Bradford, CT, USA) to the pig genome (Sus scrofa, Genbank Accession AEMK01000000) to determine the amount of non-target amplification of pig genome origin. Coverage and Shannon diversity for each library was calculated using Mothur [22 (link)]. Principal coordinates analysis of jackknifed Bray–Curtis dissimilarity matrices was performed in QIIME [23 (link)].

For the exon array analysis, the robust microarray analysis (RMA) algorithm was used for background correction, intra- and intermicroarray normalization, and expression signal calculation [19 (link)]. Significance analysis of microarray (SAM) [20 (link)] was used to calculate significant differential expression. All bioinformatic analyses were performed with the statistical program R, as previously described [21 (link)].
The expression data from quantitative SYBR Green PCR were not normally distributed, so nonparametric tests were used. Expression levels of TET2 in the different groups were analyzed using the Mann-Whitney test with a two-tailed value of P < 0.05 taken as indicating statistical significance. All tests were performed using SPSS v19.0.
Sequencing data from the Sequence Capture experiments were analyzed using GS Run Browser and GS Reference Mapper software, version 2.0.01 (Roche Diagnostics, Mannheim, Germany). All putative variants were compared with published single-nucleotide polymorphism (SNP) data (dbSNP build 130).
Amplicon deep-sequencing data were generated using GS FLX Sequencer Instrument, version 2.3, and analyzed with GS Amplicon Variant Analyzer, version 2.3 (Roche Diagnostics). The results were further processed and visualized following a previously described pipeline [17 (link)].

Paired end libraries from the genomic DNA of all the three pathotypes were separately prepared with 100 bp paired end sequenced data using Hiseq1000 (Illumina) automated sequencer (Illumina, Inc., San Diego, CA, USA). The genome sequences (100 bp on average) were aligned against the P. striiformis pathotype 78-1 (Puccinia Group Sequencing Project, Broad Institute of Harvard and MIT (http//www.broadinstitute.org)15 , using ABySS software. Reference-based assembly was performed for the processed data by GS Reference Mapper (Roche) with default parameters (minimum read length = 20 bp, minimum overlap length = 40 bp, minimum overlap identity = 90%, alignment identity score = 2, and all contig threshold = 100) with the genome sequence of P. striiformis pathotype 78-1 as the reference. Raw reads of pathotype 31, K, and 46S 119 were also mapped against the assembled data of self and other two pathotypes. The quality of the assembly was carried out by QUAST 3.2 software tool (Fig. S1, Table S1). Furthermore, the de novo assembly of the unassembled reads as well as the alignment of the raw reads of each pathotype as a whole and individually with the assembled data of their respective partner was performed using CLC Genomics Workbench 7.0. with default parameters (minimum contig = 100 bp, 23 K-mer, similarity fraction = 80% and length fraction = 50%).

Genomic DNA of B. glumae LU8093 was isolated with the Masterpure DNA purification Kit (Epicentre, Madison, USA). Genome sequencing was carried out with a hybrid approach using the 454 GS-FLX system with Titanium chemistry (Roche Life Science, Mannheim, Germany) and the Genome Analyzer IIx (Illumina, San Diego, CA). Sequencing results in 437,363 and 3,998,786 reads, respectively. In order to identify SNPs, sequence reads of LU8093 were mapped onto the B. glumae PG1 reference genome (Voget et al. 2015 (link)) with the GS Reference Mapper (Roche Life Science, Mannheim, Germany). All candidate SNP positions were then manually verified by PCR-amplifying corresponding genome regions and re-sequencing these fragments. Manual editing steps were performed using the GAP4 software package v4.6 (Staden 1996 (link)).

Five hundred nanograms of the cDNA was processed for sequencing using Roche’s GS FLX Titanium chemistry following Roche’s Rapid Library Preparation and emPCR Lib-L method manual. The library was sequenced on Roche’s GS Junior sequencing system as per manufacturer’s instructions. Using Roche’s GS Reference Mapper, the sequence data obtained was used to perform a reference-guided alignment using the default parameters with the exception of the minimal overlap identity modified from 90% to 40%.

The libraries were pooled in equimolar amounts and combined with capture beads at a ratio of two molecules from each DNA library per capture bead. The pooled DNA was then amplified with emulsion PCR (emPCR). The bead-attached DNAs were denatured, eluted, and quantified with the provided bead counter. All were performed according to the GS Junior Titanium Series emPCR (Lib-L) Manual (June 2012).
A total of 500,000 enriched DNA beads were mixed with Packing Beads. Then, the Pico Titer Plate was sequentially loaded with Prelayer Beads, DNA-Packing Beads, Postlayer Beads, and PPiase Beads. Finally, the Pico Titer Plate was mounted in the 454 GS Junior Sequencer, and the program was run in full processing mode for shotgun sequencing, according to the GS Junior Titanium Series Sequencing Method Manual (June 2012). The resulting reads were aligned, and variants were compared to the reference genome with the 454 integrated software (GS Reference Mapper; Roche, Pleasanton, CA, USA).