Trueseq dna sample prep kit

For WGS datasets, two separate library preparation protocols were used. The gDNA libraries for full genome libraries were prepared using the reagents from a TrueSeq DNA Sample Prep Kit according to the manufacturer’s instructions (TrueSeq DNA Sample Preparation Guide, revision C; Illumina Inc., San Diego, CA) with minor modifications. After the ligation, the first protocol uses a gel-free method for samples instead of a gel step that was used for the second protocol. Furthermore, the number of PCR cycles in the PCR enrichment step differs between the two protocols (five and ten cycles, respectively). A High Sensitivity DNA chip (Agilent Technologies 2100; Santa Clara, CA) was used for quantification and samples were subsequently sequenced on an Illumina HiSeq 2000 sequencer at the same laboratory.
Libraries for the WES samples were prepared using the Agilent SureSelect Kit (Agilent Technologies, Santa Clara, CA), Nimblegen Capture Kit V2 or Nimblegen Capture Kit V3 (Roche NimbleGen Inc., Madison, WI), according to the manufacturer’s instructions. A High Sensitivity DNA chip (Agilent Technologies 2100) was used for the quantification and the samples were subsequently sequenced on an Illumina HiSeq 2000 sequencer at the same laboratory.
The library preparation and sequencing of all RNA-Seq samples are described elsewhere [23 (link),24 (link)].

TrueSeq DNA Sample Preparation Kit (Illumina) was used to prepare the WGS library from purified genomic DNA sample of Comp1 strain. The quality and concentration of isolated DNA was assessed for OD₂₆₀/OD₂₈₀ ratio > 1.8, OD₂₆₀/OD₂₃₀ ratio > 1.9, DNA concentration between 250 to 500 ng/μl and no visible evidence of DNA degradation or contamination with RNA. Approximately 1.5 μg of high quality genomic DNA was used to generate fragments of size 300–400 bp by Covaris. Fragments were end-repaired by mixing with End Repair Mix and purified by Ampure XP reagent (Beckman Coulter). These fragments were adenylated and ligated to DNA Adapter Indexes for multiplexing with DNA Ligase Mix. The ligation products were purified and were subsequently enriched by PCR amplification with PCR Master Mix (TrueSeq DNA Sample Prep Kit, Illumina) according to manufacturer’s recommended protocol. The quality and quantity of the genomic DNA library thus obtained was assessed by 2100 Bioanalyzer (Agilent) and real time PCR with Kapa Library Quant Kit (Kapa Biosystems) in ABI 7900HT system (Life Technologies). Genomic DNA library of fragment size between 400 to 500 bp was selected and sequenced on the HiSeq-2000 System (Illumina) using TrueSeq PE Cluster Kit v3 and TrueSeq SBS Kit v3 (Illumina).

Total microbial genomic DNA was extracted using the QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany). The quality and quantity of extracted DNA were evaluated using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and agarose gel electrophoresis. Metagenome library preparation was conducted using a TrueSeq DNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instruction. The quantity of each library was measured fluorometrically using the Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Metagenome sequencing was performed at Novogene Inc. (Nanjing, China) using an Illumina NovaSeq 6000 Sequencing System with 150 bp paired-end sequencing of ~350 bp insert fragment.