Hiseq 2000 sequencing

DNA extracted from blood samples was available for the two Finnish cattle breeds (Eastern Finncattle and Western Finncattle) and one Siberian breed (Yakutian cattle) from a previous study (Li et al., 2007 (link)). Five unrelated individuals from each breed (14 females and one Yakutian cattle bull) were examined. Genomic DNA was extracted using a standard phenol/chloroform-based protocol (Malke, 1990 (link)). For sequencing library preparation following the manufacturer’s specifications, the genomic DNA of each individual was fragmented randomly. After electrophoresis, DNA fragments of desired length were gel purified. One type of library was constructed for each sample (500 bp insert size); 15 paired-end DNA libraries were constructed for the 15 samples. Adapter ligation and DNA cluster preparation were performed, and the DNA was subjected to Illumina HiSeq 2000 sequencing using the 2 × 100 bp mode at Beijing Genomics Institute (BGI, Shenzhen, China). Finally, paired-end sequence data were generated. To ensure quality, the raw data was modified by the following two steps using SOAPnuke (Chen et al., 2018a (link),b (link)): first, the contaminating adapter sequences from the reads were deleted, and then, the reads that contained more than 50% low-quality bases (quality value ≤5) were removed.

Blood was collected from the jugular vein of the experimental animals, and a blood genomic DNA extraction kit (DP348–03) and a high-throughput magnetic bead extraction system were used to extract the genomic DNA from the blood samples. The DNA obtained was subjected to Illumina HiSeq 2000 sequencing (Beijing Nuohe Zhiyuan Biological Information Technology Co., Ltd.).

DNA was extracted from the cow hindgut, manure, and anaerobic digester biomass samples using the PowerSoil DNA isolation kit (MoBio, Carlsbad, CA, USA). DNA extracted from the cow hindgut biomass samples was sent to the Earth Microbiome Project at the University of Colorado Boulder for further sample processing (i.e., PCR amplification via universal primers targeting the V4 region of the 16S rRNA gene—515F forward primer and 806R reverse primer—amplicon cleanup, and Illumina HiSeq 2000 sequencing). Details of the sample processing can be found at www.earthmicrobiome.org (50 (link)).
For the cow manure and anaerobic digester samples, we employed a modified version of the Earth Microbiome Project protocol (50 (link)). The modified protocol was outlined previously by Regueiro et al. (51 (link)), with the exception that in this study, 30 PCR cycles were used instead of 25. As in the study by Regueiro et al. (51 (link)), we performed duplicate PCRs of the extracted DNA samples and pooled the resulting amplicons prior to sequencing. Samples were sent for paired-end sequencing (2 × 250 bp) on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) at the Cornell University Biotechnology Resource Center (Ithaca, NY, USA).

All 123 Proteus strains in this study were screened for the presence of SXT/R391-like ICEs using a PCR-based method targeting the int_SXT gene18 (link), which encodes a conserved SXT/R391 ICE integrase. Next-generation sequencing (NGS) was performed with the PCR-positive isolates. Genomic DNA was extracted from 5 ml of overnight cultures using a Wizard Genomic DNA Purification kit (Promega, USA) according to the manufacturer’s instructions. The extracted DNA was dissolved in Tris-EDTA buffer and stored at −20 °C prior to sequencing. The genomes were commercially sequenced using Illumina HiSeq 2000 sequencing (Illumina Inc., San Diego, CA, USA) by constructing two paired-end libraries with average insert lengths of 500 bp and 2000 bp. Then, 100× libraries were obtained with clean paired-end read data. Assembly was performed using SOAP denovo v2.0419 (link).

Small RNA library was constructed as described [16 (link)]. In brief, low-molecular-weight RNAs were enriched using the Small RNA Sample Pre Kit (Illumina, San Diego, CA, USA) and sequentially ligated a 3′ adapter and a 5′ adapter. The final purified ligation products were reverse transcribed into cDNA using SuperScript^TM III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The first-strand cDNA was then PCR amplified and subjected to Illumina Hiseq2000 sequencing (Novogene, Beijing, China). The raw reads were filtered by trimming adapter sequences and removal of poly A/T/C/G reads and low-quality reads. The clean sRNA reads ranging from 18 to 26 nts in length were mapped to the cotton genome, and the clean unmapped sRNA reads were assembled using the Velvet program [17 (link)]. Assembled contigs were compared against the Genbank database using BLASTN (http://www.ncbi.nlm.nih.gov/, accessed on 15 January 2017).