Truseq dna sample prep v2 guide

Total microbial genomic DNA was extracted from the fecal samples using the PowerFecal DNA isolation kit (QIAGEN, Inc., Valencia, CA, USA), according to the manufacturer’s instructions. The DNA concentration and purity were measured using the Qubit (Thermo Fisher Scientific, Waltham, MA, USA). Agarose gel electrophoresis was performed to assess the DNA quality. The DNA samples that met the criteria of metagenomic sequencing were used for library preparation: (i) DNA concentration of >15 ng/µL; (ii) total amount of DNA >6 µg; (iii) non-contaminated and intact DNA fragment.
Shotgun metagenomic DNA libraries were constructed based on the Illumina TruSeq DNA Sample Prep V2 Guide (Illumina, Inc.; San Diego, CA, USA), with shearing to 300- to 400-bp fragments. Shotgun metagenomic sequencing was performed on Illumina platform using paired-end 2 × 150 bp chemistry (Novogene, Beijing, China). Ultimately, we obtained gut metagenome data via shotgun metagenomic sequencing from seven wild giant pandas, five wild red pandas, seven captive giant pandas, and five captive red pandas.

The metagenomic DNA libraries were constructed according to the Illumina TruSeq DNA sample prep v2 guide. The library insert sizes were checked using a DNA LabChip 1000 kit on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All libraries were sequenced on a HiSeq 2000 instrument with the PE100 mode (Illumina, San Diego, CA, USA).

The metagenomic DNA libraries were constructed with 2 μg genome DNA according to the Illumina TruSeq DNA Sample Prep v2 Guide, with an average of 350 bp insert size. The quality of all libraries was evaluated using an Agilent bioanalyser with a DNA LabChip 1000 kit. Sequencing was performed by Illumina Hiseq2500 at WuXi AppTec of China.

Following the Illumina TruSeq DNA Sample Prep v2 Guide (Illumina, Inc., San Diego, CA, USA), we constructed the DNA paired-end libraries with an insert size of 500 bp for the 76 fecal samples (24 from untreated active BD patients and 52 from normal controls). The quality of all libraries was evaluated using an Agilent bioanalyzer (Agilent Technologies, Wokingham, UK) and the DNA LabChip 1000 kit. All samples were subject to 150 bp paired-end sequencing on an Illumina HiSeq 4000 platform (Illumina, Inc., San Diego, CA, USA).
Raw reads were filtered to trim nucleotides from the 3′ end using a quality threshold of 30 and remove adaptor contamination and low-quality reads (e.g., reads containing more than 50% nucleotides below Q30, reads short than 70 bp, and reads mapped to the human genome based on alignment with SOAPaligner 2.21 [28 (link)]). As a consequence, an average of 95.82% high quality reads was obtained from all samples.

For Illumina library preparation, genomic DNA was sheared to an average 500-bp fragment length using a Bioruptar ultrasonicator. Following the Illumina TruSeq DNA Sample Prep v2 Guide (Illumina, Inc., San Diego, CA, USA), DNA paired-end libraries were constructed for the 225 fecal samples (71 from CRC patients, 63 from colorectal adenoma patients, and 91 from normal controls). The quality of all libraries was evaluated using an Agilent bioanalyzer (Agilent Technologies, Wokingham, UK) and the DNA LabChip 1000 kit. All samples were subject to 150 bp paired-end sequencing on an Illumina HiSeq platform (Illumina, Inc., San Diego, CA, USA).
Raw reads were pre-processed for 3’ end trimming using a quality threshold of 30 and filtered to exclude adaptor contaminated reads and low-quality reads (e.g., reads containing more than 50% nucleotides below Q30, reads short than 70 bp, and reads mapped to the human genome based on alignment with SOAPaligner 2.21) (Li et al., 2008 (link)). Consequently, an average of 93.3% of high-quality reads (defined as clean data) was obtained from all samples.

The DNA library was constructed following the manufacturer’s instructions (Illumina). Briefly, the DNA was sheared using an M220 Focused-ultrasonicator^TM (Covaris Inc., Woburn, MA, USA), and fragments of ~300 bp were extracted for paired-end library construction. The DNA fragments were processed by end repair, A-tailing, adapter ligation, DNA size selection, PCR, and PCR-product purification according to the Illumina TruSeq DNA sample prep v2 guide. The average insert size of the library was 350 bp. Paired-end sequencing (2 × 100 bp) was performed on an Illumina genome analyzer (HiSeq2000, Illumina) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) using the TruSeq PE cluster kit v3-cBot-HS and the TruSeq SBS kit v3-HS according to the manufacturer’s instructions (Illumina). All original metagenomic sequences were archived at the NCBI Sequence Read Archive (SRA) under accession SRP140747.