Truseq nano dna ht library preparation kit

The whole genome sequence of A. variabilis NCCPF 102052 was performed using paired-end (PE)/(MP) 2*150/2*125 base pair library on Hiseq platform (Illumina, San Diego, California, USA). The paired-end sequencing library was prepared using Illumina TruSeq Nano DNA HT library preparation kit and the mean size of the library was 645 bp. The Illumina mate- pair libraries were prepared using Cre-Lox recombination method with insert size of 3-5Kb [42 (link)]. The raw data was filtered using trimmomatic v0.30 [43 (link)] and per base sequence quality score (Q) ≥20 was considered. High quality data were assembled using Soapdenovo2 assembler as described by Li et al., to generate scaffolds using optimized parameters [44 (link), 45 ]. The whole genome sequence of Apophysomyces trapeziformis (B9324, GenBank accession ID: JNDP00000000.1) and Apophysomyces elegans (B7760, GenBank accession ID: JNDQ00000000.1) were downloaded from NCBI genome database for the comparative analysis.

Total genomic DNA was extracted, and whole genome sequencing of the M7 mutant was performed using Illumina platform at M/S Xcelris Genomics, Ahmedabad, India. Illumina TruSeq Nano DNA HT Library Preparation Kit was used for paired end sequencing library preparation. The generated libraries were sequenced on Illumina Nextseq 500 using 2 × 150 bp chemistry. T. virens wild-type sequence (LQCH00000000) was used as a reference genome for M7 genome mapping using a Burrows-Wheeler Aligner (BWA) (v. 0.7.5a) program with optimized mapping parameters. The genome coverage, gene prediction, and total number of single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (INDELs) were analyzed and compared with the reference genome. Absence of the genes in the deleted regions was further confirmed by PCR amplification using gene specific primers (Supplementary Table S1) of representative genes using the following conditions: denaturation at 95°C for 5 min, annealing at 57°C for 45 s, extension at 72°C for 1 min, and 30 cycles.

Genomic DNA was either extracted fresh from tissue (fin clips or muscle) preserved in ethanol at -20 °C or an archive of purified DNA. Fresh extracts were prepared from 20 mg tissue with DNeasy Blood and Tissue Kit (Qiagen), while archived DNA samples were originally extracted using various nucleic acid purification methods (salt extraction, Promega kit, phenol-chloroform). DNAs from both sources were screened using agarose gel electrophoresis (1%) and fluorometric quantification (Quantus dsDNA One, Promega). DNA samples were ascribed integrity scores ranging from 0 (completely degraded/absent) to 5 (high molecular weight) and included in the analysis if at least a faint band of genomic DNA was observed in addition to a smear. Genomic DNAs were maintained at -20 °C until use.
Sequencing libraries were prepared using the TruSeq Nano DNA HT Library Preparation Kit (Illumina) following the manufacturer’s protocol in a 96-well plate format. Library integrity was validated on the QIAxcel capillary electrophoresis system (Qiagen) using a high-resolution gel cartridge and the OM500 analysis method with 15 bp-3Kb alignment marker and a 100 bp-2.5 kb size/concentration standard (Fig. S1, Supporting Information) and were quantified with fluorometric assays (Quantus sDNA One, Promega or Qubit).

Indexed DNA library for NGS was prepared using the TruSeq® Nano DNA HT Library Preparation Kit (Illumina, cat. 20,015,965) according to manufacturer’s recommendation. Indexed libraries were then quantified using a Q-bit fluorometer (Thermo Fisher Scientific) and pooled together at an equal concentration. The pooled library was diluted to 18 pM concentration. Sequencing was performed on an Illumina MiSeq® using 2 × 300 PE chemistry according to manufacturer’s protocol. The reads were de-multiplexed with the MiSeq Reporter software and were stored as FASTQ files for downstream analysis (Additional file 14: Table S7).

A paired-end sequencing library was prepared using an Illumina TruSeq Nano DNA HT Library Preparation Kit (Kumar et al., 2017 (link)). A 100-ng sample of g-DNA for the 350-bp insert size (2 libraries) and a 200 ng sample of g-DNA for the 550-bp insert size were fragmented with a Covaris instrument according to the manufacturer’s instructions. The covariance program for the 350-bp insert size and 550-bp insert size was calculated.
A mate-pair sequencing library was prepared using the Illumina Nextera Mate-Pair Sample Preparation Kit (Campbell et al., 2016 (link)). 1 μg of the gDNA was simultaneously fragmented and tagged with a biotinylated mate-pair junction adapter. An AMPure bead purification step was performed to clean up the PCR product and remove the smallest fragments (<300 bp) from the final library. Quantity and quality checks (QCs) of the library were performed in a Bioanalyzer 2100 instrument (Agilent Technologies) using a high-sensitivity (HS) DNA chip.