Genome analyzer 2 system

Whole-genome sequencing was conducted by Illumina Genome Analyzer II system, with insert length of about 500 bp. Total 6,060,140, 1,904,646, 2,474,758 and 13,680,640 reads with average lengths of 60 to 91 bp were obtained from F. fructosus NRIC 1058^T, F. ficulneus JCM 12225^T, F. pseudoficulneus DSM 15468^T and F. tropaeoli F214-1^T, respectively. De novo assembly using the Velvet Assembler for short reads with parameters optimized by the VelvetOptimizer (Version 1.2.10) [14 (link)] resulted in 57, 28, 15 and 101 contigs each (Length: 1,489,862, 1,552,198, 1,413,733 and 1,686,944 bp; N₅₀: 89,458, 226,528, 283,981 and 226,443 bp). The k-mer sizes for the strains were 81, 45, 51, 63 bp each. The genome was annotated using the Microbial Genome Annotation Pipeline (MiGAP) [15 ] with manual verification. In the pipeline, protein coding sequences (CDSs) were predicted by MetaGeneAnnotator 1.0 [16 (link)], tRNAs were predicted by tRNAscan-SE 1.23 [17 (link)], rRNAs were predicted by RNAmmer 1.2 [18 (link)], and functional annotation was finally performed based on homology searches against the RefSeq, TrEMBL, and Clusters of Orthologous Groups (COG) protein databases.

To ensure unbiased analysis of tissue response, the total RNA was isolated from random sections (10–15 mg) of liver. The RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using a RNeasy Plus Mini kit (Qiagen, Mississauga, ON, Canada). Three livers of each group were sequenced, and the data were analyzed. In brief, the RNA was sequenced on one sequencing lane of an Illumina Genome Analyzer II system (Illumina). The paired-end reads were mapped to the mouse genome (version NCBIm37/mm9) using Tophat. The mapped reads were used to quantify the transcripts from the RefSeq reference database. The genes met the fold change > 2 and Benjamin Hochberg adjust Pvalue (B H Pvalue) < 0.05 were the significant differently expressed genes which were used for the following analysis. For the functional annotation analysis of genes, the BiNGO (http://apps.cytoscape.org/apps/bingo) was used. The GO terms met the B H Pvalue < 0.001 were the significant GO terms.

The extracted DNA was used in a combined sequencing approach using a 454 GS-FLX Titanium XL system with Titanium chemistry (Roche Life Science, Mannheim, Germany) and the Genome Analyzer II system (Illumina, San Diego, CA, USA) as recommended by the manufacturers. Resulting reads were assembled into contigs using MIRA software. For P. larvae DSM 25719 (ERIC I), shotgun sequencing resulted in 38.35-fold and 56.49-fold coverage from 454 and Illumina reads, respectively. In case of P. larvae DSM 25430 (ERIC II), an average coverage of 64.4-fold was determined (30.07-fold 454 coverage and 35.01-fold Illumina coverage). Editing of shotgun sequences and 454 sequences were done by using GAP4, as part of the Staden software package [86] (link). To solve problems with misassembled regions caused by repetitive sequences and close remaining sequence gaps, PCR reactions, combinatorial multiplex PCRs reactions, fosmid libraries, plasmid libraries, and primer walking with recombinant plasmids were used. PCR reactions have been carried out with the BioXact Kit (Qiagen, Hilden, Germany) and Phusion High Fidelity DNA Polymerase Kit (Thermo Fisher Scientific, Schwerte, Germany) as described by the manufacturers.

Sequence tags were prepared using Illumina’s Digital Gene Expression Tag Profiling Kit (San Diego, CA, USA) according to the manufacturer’s instructions. Then, sequencing was performed using the Illumina Genome Analyzer II system according to the manufacturer’s protocols. Image analyses, base calling, raw 17 bp tag generation and tag counting were performed using the Illumina Analysis Pipeline.

Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini kit. Genomic DNA (3 μg) was randomly fragmented by Covaris, and ligated with adaptors. After purification using Agencourt AMPure SPRI beads, the adaptor-ligated templates were amplified by ligation-mediated PCR and hybridized to the SureSelect Biotinylated RNA Library for enrichment. Whole Exome sequencing (WES) was performed using the Agilent SureSelect Human All Exon Capture kit and 90-bp paired-end sequencing on an Illumina Genome Analyzer II system. For verification of variants, Sanger sequencing was conducted as previously described (Olesen et al., 2012 (link)).
We trimmed raw reads and filtered low-quality reads using cutadapt vX (Martin, 2011 (link)) and prinseq vX (Schmieder and Edwards, 2011 (link)). Alignment [with Burrow-Wheelers Aligner mem algorithm (Li and Durbin, 2009 (link))] to the human reference genome (NCBI Build 37) and post-processing was made according to Genome Analysis Toolkit version 3.4 (GATK) guidelines (Auwera et al., 2013 (link)). Variant calling was performed with Haplotypecaller/GATK v3.4, followed by quality control and variant filtering (Supplementary Figure S1). Relatedness was inferred using the King-robust algorithm (Manichaikul et al., 2010 (link)).

Ribosomal RNA was digested during the construction of the library, then the fragments were randomly divided into 250–300 bp. The cDNA library was prepared using the TruSeq Double-Strand Total RNA Library Prep Kit with random primers to generate fragmented RNA for the first-strand cDNA. For the second cDNA strand, the dNTP reagent in the dTTP was replaced by Dutp, resulting in the incorporation of A/U/C/G bases into the second cDNA strand. Following end repair and addition of poly-A tails, cDNA fragments of 150–200 bases were isolated, and single-stranded cDNA was obtained using uracil aza-glycosylase (UNG). Then, nine RNA-Seq libraries were generated. The data are publicly available in the NCBI GEO database with the accession number PRJNA867525. The sequencing was conducted on the Illumina Genome Analyzer II System at Beijing Novo-gene Bioinformatics Technology Co., Ltd. (Beijing, China), utilizing paired-end high-throughput sequencing.

In order to analyze the effect of mouse liver total transcriptome after MBCD treatment, 15 mg liver was collected to isolate the total RNA using RNAiso Plus reagent (Takara Biochemicals, USA) according to the manufacture’s protocol. Three livers of C and H group were sequenced, and the data were analyzed. The RNA sequencing was performed at Wuhan MetWare Biotechnology Co., Ltd. (www.metware.cn) using Illumina Genome Analyzer II system (Illumina). Functional annotation analysis of differential genes was performed using the Annotation, Visualization and Integrated Discovery, (DAVID, https://david.ncifcrf.gov/home.jsp).