Truseq dna pcr free lt library prep kit

Genomic DNAs were extracted from dried mycelia of two homokaryons (BS05 and BS17) and a heterokaryon (BS05 × BS17) by using a SLA-32 automatic nucleic acid extractor with a TANBead^® Fungi DNA extraction kit. Approximately 1 μg of a genomic DNA was sonicated to ~350 bp with the Covaris M220 system using the following parameters: duty factor 20%, peak/display power 50 W, 200 cycles per burst, 65 s, 20 °C. The sonicated DNA was purified, end-repaired, A-tailed and ligated with adaptors according to the manufacture’s guide (Illumina Truseq DNA PCR-Free LT library prep kit) followed by purification and sequencing. A paired-end DNA-seq library was sequenced using Illumina HiSeq 2500 platform per manufacturer’s instructions. Sequencing was performed up to 250 cycles. Image analysis and base calling were performed with the standard Illumina pipeline. In total 17,621,957, 23,362,228, and 18,348,369 reads were aligned to the reference genome Termitomyces sp. J132 (Poulsen et al. 2014 (link)), with the estimated depths of 32.3×, 42.8×, and 33.6× for BS05 × BS17, BS05, and BS17, respectively.

DNA was extracted from samples using the PowerMax Soil DNA Isolation Kit according to manufacturer’s instructions, with minor modifications (see Additional file 1: Supplementary Methods). Library preparation of Metagenome and Resistome libraries followed standard commercial kit protocols, with some modifications. For the Metagenome libraries, the TruSeq DNA PCR-Free LT Library Prep Kit (Illumina) was used, while resistome libraries were created using the SureSelect^XT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (Agilent Technologies) with the custom-designed MEGaRICH bait set. The Resistome-UMI and Metagenome-UMI libraries were created using a protocol that incorporated dual-indexed UMI adapters (see Additional file 13) into sequence libraries [23 (link)], with modifications to allow for integration with Agilent SureSelect protocols (see Additional file 1: Supplementary Methods for details).

Before library preparation, genomic DNA was fragmented to an average insert size of 350 nt using 18 cycles of 30 s each on a Bioruptor Diagenode sonication device, split into three groups of 6 cycles with 2.5 min on ice between each set. The genomic DNA sequencing libraries were prepared using the TruSeq DNA PCR-Free LT Library Prep Kit following the manufacturer’s instructions (Illumina, San Diego, CA), starting from 1.1 µg of sonicated DNA. Libraries were validated using High Sensitivity D1000 screentape on the 2200 Tapestation instrument (Agilent technologies, Santa Clara, CA) and the LightCycler 480 Instrument II using the LightCycler 480 SYBR Green I Master mix (Roche, Basel, Switzerland).

The genomic insertion sites for P elements were amplified according to the protocol of Tsukiyama et al. [50 (link)] with minor modifications. The genomic DNA extracted from 40 adult flies was digested with HhaI or TaqI (TaKaRa, Japan) and ligated to overhanging adapters. Using these ligation products as a template, PCR was performed with primers specific to the adaptor and to P elements respectively. Nested PCR was performed to specifically amplify P-element-containing PCR fragments. About 300- to 600-bp-long HhaI and TaqI products were purified from an agarose gel, and used for preparation of deep sequencing libraries with the TruSeq DNA PCR-Free LT Library Prep Kit (Illumina, California, USA). Pair-end 250-bp sequencing was performed on the MiSeq system (Illumina). Details are shown in the Additional file 1.

For comparison of exome capture technologies with conventional WGS approach, we used several recent samples sequenced at Biobank genome facility²⁷ . WGS libraries were prepared using TruSeq DNA PCR-Free LT Library Prep Kit (Illumina, USA) according to the manufacturer’s protocol. Additionally we used PCR-free WGS data of the Genome In A Bottle (GIAB) consortium²⁸ (Chinese and Ashkenazi trios), as well as several samples publicly available at the NCBI Sequencing Read Archive (SRA) (SRA IDs SRR2098244, SRR2969967, ERR2186302, SRX2798634, SRX2798624). For GIAB samples, we used pre-calculated Novoalign BAM files available at the GIAB FTP site (ftp://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data/). For our own WGS samples and samples downloaded from SRA, we used bwa mem v0.7.1 for read alignment. All BAM files were narrowed down to the GENCODE v19 CDS regions using bedtools^{29 (link)}. We further downsampled the 300x BAM file for GIAB sample HG001 to obtain 5 separate BAM files with 60x mean coverage or 10 BAM files with 30x mean coverage (Fig. 5).