Viia 7 real time thermocycler

For each stool sample, a sequencing library was first constructed using Illumina’s Truseq Nano DNA Library Preparation Kit (Illumina, San Diego, USA). The samples were sheared on a Covaris E220 to ~ 450 bp, following the manufacturer’s recommendation, and uniquely tagged with one of Illumina’s TruSeq HT DNA barcode combinations to enable sample pooling for sequencing.
The finished libraries were quantitated using Invitrogen’s Picogreen assay and the average library size was determined on Bioanalyzer 2100, using a DNA 7500 chip (Agilent). Library concentrations were then normalized to 4 nM and validated by qPCR on a ViiA-7 real-time thermocycler (Applied Biosystems), using qPCR primers recommended in Illumina’s qPCR protocol, and Illumina’s PhiX control library as standard. The libraries were then pooled at equimolar concentrations and sequenced on an Illumina HiSeq2500 sequencer in rapid mode at a read-length of 250 bp paired-end. Approximately 5Gb of sequencing data were obtained per sample so as to capture most of the novelty [19 (link)].
Genomic DNA sequences obtained from Illumina HiSeq paired-end sequencing were analyzed as follows: (i) Sequence quality check, (ii) Reads to protein alignment, and (iii) Taxonomical classification. To perform a quality check, reads shorter than 30 bp and low-quality sequences (Therefore, removal of any sequence with a higher error rate than 1% (i.e., Thereafter, metagenomic reads were obtained by aligning the high-quality reads against human reference genome (hg19), whereby reads that were aligned to hg19 were discarded. The non-human metagenomic reads were then run on ResFinder 2.1. to check for the ARG’s conferring resistance to: aminoglycoside, tetracycline, beta-lactam, colistin, fosfomycin, fusidic acid, macrolide, nitroimidazole, oxazolidinone, phenicol, quinolone, rifampicin, sulphonamide, trimethoprim, and glycopeptide antibiotics.
Lastly, a lowest common ancestor (LCA)-based Taxonomical Classification of the aligned sequence reads was carried out on MEGAN6 (MEtaGenome Analyzer 6) using a bitscore cut-off of 100. A simple algorithm is utilized by MEGAN to assign each read to the LCA of the set of taxa that it hits in the comparison, whereby species-specific sequence reads will be assigned to the species taxon, while widely conserved sequence reads will be assigned to the high-order taxa [20 ].

The quality of the RNA samples was determined by running the samples on a Bioanalyzer RNA 6000 Pico Chip (Agilent). Next-generation sequencing library preparation was performed following Illumina’s TruSeq Stranded mRNA Sample Preparation protocol with the following modifications: RNA samples were added to the elute-fragment-prime step. The PCR amplification step, which selectively enriches for library fragments that have adapters ligated on both ends, was performed according to the manufacturer’s recommendation. Each library was uniquely tagged with one of Illumina’s TruSeq LT RNA barcodes to allow library pooling for sequencing. Library quantitation was performed using Invitrogen’s Picogreen assay and the average library size was determined by running the libraries on a Bioanalyzer DNA 1000 chip (Agilent). Library concentrations were normalized to 2 nM and validated by qPCR on a ViiA-7 real-time thermocycler (Applied Biosystems), using qPCR primers recommended in Illumina’s qPCR protocol, and Illumina’s PhiX control library as standard. Libraries were then pooled and sequenced in one lane of an Illumina HiSeq2500 rapid sequencing run at a read-length of 101bp paired-end. Sequencing data have been submitted to GenBank SRA archive with the BioProject ID: PRJNA286974 and SRP accession SRP059483.