Truseq nano dna lt sample preparation kit

Genomic DNA was extracted with Easy-DNA gDNA purification Kit (Life Technologies, Carlsbad, CA) from 6 ml of 311 betaine cultures of methanol-adapted S. ovata strains met-T4-2, met-T9-1, met-T18-2, and met-T18-3. The genomic libraries were generated with the TruSeq Nano DNA LT Sample Preparation Kit (Illumina Inc., San Diego CA). Briefly, 100 ng of genomic DNA diluted in 52.5 μl TE buffer was fragmented with a Covaris E220 ultrasonicator (Woburn, MA). The ends of fragmented DNA were repaired by T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase. The Klenow exo minus enzyme was then used to add an ‘A’ base to the 3′ end of the DNA fragments. After ligation of the adapters, DNA fragments ranging from 300 to 400 bp were recovered by beads purification. The adapter-modified DNA fragments were enriched by three cycles-PCR. The average size of dsDNA fragments in the libraries was determined with an Agilent 2100 Bioanalyzer. Sequencing was done with a MiSeq Reagent kit v2 (300 cycles) on a MiSeq (Illumina) platform with a paired-end protocol and read lengths of 151 nucleotides. The sequencing reads were then trimmed with Trimmomatic64 (link) before being used for variant calling with breseq65 . The reference genome for the analysis was Sporomusa ovata DSM 2662 (NCBI accession ASXP00000000.1). All the samples had an average coverage of at least 30X.

For whole genome re-sequencing, pools of male and female adult worms (n = 50) were made for each strain of H. contortus. Total genomic DNA from each pool sample was isolated through Qiagen DNeasy Blood & Tissue Kit, Hilden, Germany (cat no. 69506), according to its protocol. DNA samples were fragmented using Covaris’ Focused-ultrasonicator S220. Libraries were constructed using TruSeq Nano DNA LT Sample Preparation Kit, San Diego, CA, USA (Illumina, Cat.No.FC-121-4001). Briefly, the DNA fragments were end-repaired, adenylated at 3’ end, and subjected to adapter ligations. The fragments were than enriched, normalized, and finally subjected to paired-end sequencing at Illumina HiSeqXTen platform.

Multiplex PCR was performed as previously described [35 (link)]. Multiplex PCR products were prepared using a TruSeq Nano DNA LT sample preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The samples were mechanically sheared using an M220 focused ultrasonicator (Covaris, Woburn, MA, USA). The cDNA amplicon was size-selected, A-tailed, ligated with indexes and adaptors, and enriched. The libraries were sequenced using a MiSeq benchtop sequencer (Illumina) with 2 × 150 bp with a MiSeq reagent V2 (Illumina).

Total DNA extraction from feces of all samples was performed using an Agencourt AMPure XP (Beckman Coulter, Brea, CA) kit according to the manufacturer’s instructions. The concentration and purity of extracted DNA were determined by use of a NanoDrop 2000 UV-visible (UV-Vis) spectrophotometer (Thermo Scientific, Wilmington, DE). The quality of DNA was checked by 1% agarose gel electrophoresis. Extracted DNA (1 μg in a total volume of 52.5 μL) was added to a Covaris tube and then fragmented using a DNA shearing instrument (Gene, Shanghai, China). The fragmented DNA was used to prepare a DNA library according to the protocol of TruSeq Nano DNA LT sample preparation kit (Illumina, San Diego, CA). Then, the bridge PCR was performed. The PCR amplification mixture consisted of 25 μL of purified ligation DNA, 20 μL of enhanced PCR mix, and 5 μL of PCR primer cocktail. PCR amplification conditions were as follows: 95°C for 3 min, 8 cycles of 98°C for 20 s, 60°C for 15 s, and 72°C for 30 s, 72°C for 5 min, and a 4°C hold.

According to the manufacturer’s instructions, the total DNA was isolated from the sample using QIAamp Fast DNA Mini Kit (QIAGEN, Hilden, Germany). The DNA concentration and integrity were evaluated by NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis. The DNA was fragmented with S220 Focused-ultrasonicators (Covaris, USA) and purified with Agencourt AMPure XP magnetic beads (Beckman Coulter Co, USA). Then, the library was constructed using the TruSeq Nano DNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The sequencing was performed using Illumina NovaSeq 6000.

DNA was extracted from nasal mucus samples through a MagPure Soil DNA KF Kit (Magen Biotechnology, Guangzhou, China). The DNA integrity and concentration were evaluated by an agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), respectively. DNA was then fragmented by S220 Focused-ultrasonicators (Covaris, Woburn, Massachusetts, USA) and cleaned up by Agencourt AMPure XP beads (Beckman Coulter Co., Indianapolis, Indiana, USA). Then the library construction was conducted by utilizing a TruSeq Nano DNA LT Sample Preparation Kit (Illumina, San Diego, California, USA) following the manufacturer’s instructions. A metagenomic sequencing approach was performed using an Illumina Novaseq 6000 platform (Illumina, San Diego, California, USA), and 150 bp paired-end reads were yielded.