Computational protocol: Alteration of the gut microbiota in Chinese population with chronic kidney disease

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Protocol publication

[…] Isolated fecal DNA was used as a template for amplification of the 16S rRNA V4-6 region using the universal primer V4F (5′-GTGCCAGCMGCCGCGGTAA-3′) and V6R (5′-ACAGCCATGCNCACCT-3′). 20 μl reaction mixture: 10 μl TaKaRa Premix Taq, 2 μl template DNA, 0.5 μl 10 μM barcode forward primer, 0.5 μl 10 μM reverse primer, and 7 μl double-distilled H2O. The PCR cycle conditions: an initial denaturation at 94 °C for 5 min, 25 cycles of 94 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 5 min. PCR products were sequenced using Illumina GAII (Illumina, San Diego, CA, USA) at the Beijing Genomic Institute (Shenzhen, China). Sequencing results were clustered by lllumina paired barcoded - sequencing (end) (BIPES) (PE) process for preliminary analysis, the rest of the sequence were screened by UCHIME and removed the suspected chimeric sequence. All reads were sorted into different samples according to their barcodes. Then the two stage clustering (TSC) was used for clustering to extract the OUT in order to to distinguish the high abundance and low abundance sequences. Principal coordinates analysis (PcoA) based on UniFrac distance was performed with QIIME. The linear discriminant analysis (LDA) with effect size measurements (LEfSe) were used to identify indicator bacterial groups specialized within the two groups. […]

Pipeline specifications

Software tools UCHIME, UniFrac, QIIME, LEfSe
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Homo sapiens, Bacteria, Faecalibacterium prausnitzii
Diseases Kidney Failure, Chronic, Renal Insufficiency, Chronic
Chemicals Creatinine