Base calling pipeline

Genomic DNA from the middle intestinal tissues was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and was subjected to reduced representation bisulfite sequencing (RRBS), as previously described (24 (link)). Raw sequencing data were processed by the Illumina base-calling pipeline. Low-quality reads that contained more than 30% N's or >10% of the sequence with low-quality value (quality value <20) per read were omitted from the data analysis. Bisulfite sequence mapping program was used for sequence alignment to the Ensembl pig reference genome (Sscrofa10.2). Methylation level of individual cytosine was calculated as the ratio of sequenced depth of methylated cytosine to the total sequenced depth of the individual cytosine. One of 80 samples failed in RRBS and was excluded in DNA methylation analysis.

Briefly, genomic DNA was isolated from flash frozen muscular tissue. Then, the construction of RRBS libraries and paired-end sequencing using Illumina HisSeq analyzer was performed at Novogene technology co., LTD (Beijing, China). Raw sequencing data were processed by an Illumina base-calling pipeline. Genomic DNA was digested with MspI enzyme at 37 °C for 16 h. The DNA fragments after enzyme digestion were repaired at the end, and the sequencing adapters with all cytosine methylated were attached. The inserted DNA fragments with the length ranging from 40 to 220 bp were selected for glue cutting. Then, Bisulfite conversion was carried out. After that, the unmethylated C was changed to U (after PCR amplification to T), while the methylated C remained unchanged. Finally, PCR amplification was carried out to obtain the final DNA library. Clean reads were obtained from the raw data after removing reads containing adaptor sequences, unknown, or low-quality bases. The process of quality control was carried out using Trimmomatic software [41 (link)]. Quality control was adopted to access the high data quality by (1) removing low-quality reads using a sliding window method (SLIDINGWINDOW: 4:15); (2) removing reads including adaptor sequences (ILLUMINACLIP: adapter.fa: 2:30:7:1: true); (3) removing reads with tail quality lower than 3 or with unknown bases (TRAILING: 3).

The Illumina base-calling pipeline was used for fluorescent image deconvolution, quality value calculation, and sequence conversion to obtain reads with a length of 50 nt. High-quality (clean) reads were obtained after trimming the 5′ and 3′ adaptors and eliminating contaminants and inadequate (<18 nt) and low-quality reads. The clean reads were then mapped to the mouse genome (mm9) using SOAP2 [34 (link)]. Perfectly matched reads were summarized and retained for further analyses. Read annotations were performed as described previously. Briefly, a hierarchical order that classified reads into specific RNA species was determined for annotation using the BLASTn (ftp://ftp.ncbi.nih.gov/blast/) program. The annotation order was miRNA > rRNA/snoRNA/tRNA/scRNA/snRNA > piRNA > endo-siRNA.

Raw sequencing data were processed by the Illumina base-calling pipeline. Low-quality reads that contained more than 30% ‘N’s or over 10% low-quality calls (quality value < 20) were removed. Adapter contamination was removed by cutadapt (version 1.9) [17 (link)]. The clean reads were aligned to the reference genome hg19 using BSMAP (version 2.73) [18 (link)]. Conversion ratio was calculated on lambda DNA and samples with a conversion ratio lower than 99% would be considered unqualified for further analysis and corresponding libraries would rebuilt and sequenced. Only uniquely aligned reads containing the Msp I enzyme digestion sites were used for further analysis. Only CpG sites with sequencing depths ≥5 were selected as candidate sites. After bisulfite treatment, cytosines were read as “T” if unmethylated or as “C” if methylated. Methylation level of the sample was defined as the ratio of number of “C”s and the sequencing depth of the site. Differential methylation analysis on these sites were performed with ANOVA (analysis of variance) and the acquired p-values were adjusted to Q-values with Benjamini-Hochberg method [19 ]. Differential methylation sites (DMSs) were defined as sites with a Q-value lower than 0.05.

Total RNA was isolated from the biofilms using the mirVana miRNA isolation kit (Applied Biosystems). Microbial cells were lysed and RNA was extracted by Acid-Phenol: Chloroform and ethanol precipitation and eluted in nuclease-free water. Ribosomal RNA was depleted and mRNA enriched by modified capture hybridization approach. Enriched mRNA served as a template for the polyadenylation reaction and cDNA synthesis. Microbial libraries were clustered on the Illumina HiSeq platform, and 150 bp paired-end sequencing was performed. The Illumina base-calling pipeline was used to process the raw fluorescence images and call sequences. Raw reads with >10% unknown nucleotides or with >50% low quality nucleotides (quality value < 20) were discarded. Microbial transcripts were quality filtered using SolexaQA++, and aligned against the Human Oral Microbiome Database^{71 (link)} using DIAMOND.^{72 (link)} Aligned sequences were annotated to the KEGG database using Megan 6.^{73 (link)} The metagenomic sequence classifier Kraken^{74 (link)} was used along with our custom tool, kraken-biom, for taxonomic identification. Analysis and visualization of the distribution of operational taxonomic units was performed using QIIME^{75 (link)} and PhyloToAST.^{76 (link)} Bioconductor package for R, DESeq2, was used to perform differential expression analysis of the annotated microbial transcripts.

Paired-end libraries with average insert size of approximately 500 bp were constructed for each sample according to the manufacturer’s instructions (Illumina, San Diego, CA). Library quality and concentration were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and Qubit 3.0 Fluorometer (Life Technologies, CA, USA). These libraries were subjected to 2 × 100 bp paired-end (PE100) sequencing on a HiSeq2000 instrument (Illumina). A standard Illumina base-calling pipeline was used to process the raw fluorescent images and the called sequences. Read quality was evaluated using the FastQC package (www.bioinformatics.babraham.ac.uk/projects/fastqc/). For genome re-sequencing data, short-reads were trimmed 15 bp from the 3′-end according to the base quality distributions. The raw sequencing data reported in this paper have been publicly deposited in the NCBI Short Reads Archive (SRA) with accession number SRP047477.