Computational protocol: Progressive Colonization of Bacteria and Degradation of Rice Straw in the Rumen by Illumina Sequencing

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Protocol publication

[…] The rumen fluid samples collected from all time points for each cow were mixed and used for DNA extraction. A 10 ml volume of each mixed rumen fluid was centrifuged and the resulting pellet was ground in liquid nitrogen for DNA extraction with CTAB buffer. The DNA was purified in an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1). The final DNA pellets were washed with ice cold 70% ethanol and dissolved in TE buffer.The metagenomic DNA libraries from the three cows were prepared following the manufacturer’s instructions (Illumina), and the samples were then pair-end sequenced using Illumina Hiseq4000 at BGI (Shenzhen, China). A total of 119,215,228 reads with a length of 150 bp was obtained after removing the reads containing N and the adapter (SRA accession number: SRP094501). The forward sequencing reads from each sample were quality controlled with meta-qc and the cow genome (NCBI assembly: GCA_000003205.4) was used as reference to remove the reads derived from the host. A total of 19,447,862, 19,311,185, and 19,452,969 sequences remained after quality control for each cow. MetaPhlAn () was then used to analyze the bacterial composition and ShotMAP () was used to annotate with the CAZy database. [...] The samples from the Ad (0.25 g) and As (1 ml) fractions were used for DNA extraction using the bead-beating and phenol–chloroform extraction method described by . The universal primers (341F 5′-CCTAYGGGRBGCASCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′) () were used to obtain the PCR amplicons for paired-end sequencing on an Illumina MiSeq platform at Novogene (Beijing, China). The paired-end reads (SRA accession number: SRP066537) were assembled, trimmed, quality-filtered, and analyzed according to . Briefly, the paired-end reads were first assembled and trimmed with PANDAseq () to remove primers and barcodes, as well as sequences less than 400 bp, longer than 500 bp, or with ambiguous bases. The trimmed sequences were then filtered by ea-utils () to remove sequences with mean quality score values less than 30. Subsequently, the quality-filtered sequences were analyzed by QIIME for open reference OTU picking, alpha and beta diversity analysis at a sequence depth of 17,900 sequences, and taxonomic assignment based on Greengenes (; ). The functional prediction and differential characterization were conducted with PICRUSt and LEfSe (; ). […]

Pipeline specifications

Software tools MetaPhlAn, PANDAseq, ea-utils, QIIME, PICRUSt, LEfSe
Databases CAZy Greengenes
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Oryza sativa, Bos taurus