16s metagenomic sequencing library protocol

The cecum was removed from the chicken and put into the sterile tube before snap freezing in liquid nitrogen and stored at –80°C for further analysis. Multiplexed 16S rDNA libraries were prepared using standard 16S metagenomic sequencing library protocols from Illumina (San Diego, CA), which used the V3–V4 region of 16S rDNA for target amplification. The authors used Illumina Miseq (Illumina) to performed paired-end reads (300 bp) to generate ∼200,000 sequences per sample. Subsequent analysis was performed in Quantitative Insights Into Microbial Ecology based on barcode sequences, and paired-end reads were merged (minimum score <20, mismatch). Additional quality filter steps were applied to exclude short reads <230 bp and chimeras. To ensure high coverage in all samples, samples producing <35,000 reads were excluded. Sequences after processed were clustered at 97% sequence identity to generate operational taxonomic unit and each operational taxonomic unit was annotated using the Silva128 16S rRNA database (bacteria and archaea). The fastq files generated by Illumina sequencing output were uploaded in the NCBI BioProject database (BioProject ID, PRJNA595453; submission ID, SUB6687114).

The total DNA from human stool samples was extracted using the PowerMax Soil DNA Isolation Kit (MoBio, Carlsbad, CA) following the standard protocol. The quality and quantity of DNA were measured using the PicoGreen assay kit (Invitrogen, Eugene, OR) and NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA). Illumina 16S Metagenomic Sequencing Library protocols to amplify the V3 and V4 regions were used for bacterial metagenomics analysis. Ten nanograms of the extracted DNA was used as a template for PCR amplification with 16S_Amplicon_PCR Forward_Primer (5′‐TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG‐3′) and 16S_Amplicon_PCR Reverse_Primer (5′‐GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC‐3′). The final purified products were quantified using quantitative polymerase chain reaction according to the quantitative polymerase chain reaction Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified using the LabChip GX HT DNA High Sensitivity Kit (PerkinElmer, Waltham, MA). Paired‐end (2 × 300 bp) sequencing was performed using the MiSeq platform (Illumina, San Diego, CA). Taxonomic assignment was performed using UCLUST (version 1.2.22) and QIIME (version 1.8.0) against the 16S rRNA gene sequence database in Silva (release 123).

Multiplexed 16S rDNA libraries were prepared using standard 16S metagenomic sequencing library protocols from Illumina, which uses the V3–V4 region of 16S rDNA for target amplification. The authors performed paired end reads (250 bp) sequencing to generate ∼200,000 sequences/sample using the Illumina MiSeq. Subsequent analysis was done in CLC Microbial Genomics Module 2.5 (Qiagen) and R.^{21 (link)} Paired end reads were merged (mismatch cost–2, minimum score–8, gap cost–3, maximum unaligned end mismatches–0) and trimmed to the same length. Additional quality filter steps were applied to exclude short reads, sequences with poor quality scores, and chimeras. To ensure comparable high coverage in all samples, the authors excluded samples producing <35,000 reads. They did not use OTU-based enumeration of taxa due to the over-merging that occurs. Instead each unique 16S rDNA sequence was subjected to BLAST using the NCBI 16S rRNA database (Bacteria and Archaea) to identify best matches to taxa at the genus and species levels based on % identity.