Computational protocol: Rumen Bacteria Communities and Performances of Fattening Lambs with a Lower or Greater Subacute Ruminal Acidosis Risk

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Protocol publication

[…] Bacteria DNA from LSR (n = 10) and HSR (n = 10) lambs was used for 16S rRNA gene sequencing and bioinformatics analysis. Bacterial universal primers 5′-ACTCCTACGGGAGGCAGCA-3′, 5′-GGACTACHVGGGTWTCTAAT-3′ to amplify the V3-V4 region of the bacterial 16S rRNA gene and subsequently sequence the PCR products. The PCR amplicons were sequenced by using an Illumina HiSeq 2500 platform at Biomarker Technologies Co. Ltd. (Beijing, China). Overlapping paired-end reads of the DNA fragments (minimum overlap of 10 bp, maximum mismatch rate set 0.2) were merged by using FLASH v1.2.7 (). Low-quality sequences were trimmed using a minimum average quality score of 20, with a window size of 50 bp. Chimeric reads were filtered using UCHIME v4.2 software, and reads from each sample were clustered into Operational Taxonomic Units (OTUs) based on 97% sequence similarity according to the UCLUST in QIIME (; ). Representative sequences of each OTU were assigned a taxonomy by RDP Classifier and then aligned against entries in the Silva alignment database (Release 128) (). Samples were rarefied for alpha diversity calculations, including generating curves of their OTU rank, rarefaction and Shannon index. Standard Shannon and Simpson indices and richness indices including the Ace and Chao1 index were determined by using Mothur (version v.1.30). To compare the dissimilarities in bacterial communities in LSR and HSR lambs, we performed a principal coordinate analysis (PCoA) that was based on weighted UniFrac distances at OTU level. The sequencing data were deposited into the Sequence Read Archive (SRA) of NCBI and can be accessed via accession number SRP108931. […]

Pipeline specifications

Software tools UCHIME, UCLUST, QIIME, RDP Classifier, mothur, UniFrac
Application 16S rRNA-seq analysis
Organisms Escherichia coli, Ovis aries, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Butyrivibrio fibrisolvens, Bacteroidetes, Firmicutes, Bacteria
Diseases Acidosis
Chemicals Lactic Acid