Truseq nano dna ht sample prep kit

Total DNAs were extracted from cells with DNAiso Reagent (TaKaRa, Dalian, China) following the manufacturer’s instruction. The quality of isolated genomic DNA was verified by using these two methods in combination: (1) DNA degradation and contamination were monitored on 1% agarose gels. (2) DNA concentration was measured by Qubit DNA Assay Kit in Qubit 2.0 Flurometer(Life Technologies, CA, USA). A total amount of 1μg DNA per sample was used as input material for the DNA library preparations. Sequencing library was generated using Truseq Nano DNA HT Sample Prep Kit (Illumina, USA) following manufacturer’s recommendations and index codes were added to each sample. Briefly, genomic DNA sample was fragmented by sonication to a size of 350 bp. Then DNA fragments were endpolished, A-tailed, and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification. After PCR products were purified (AMPure XP system), libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified by real-time PCR (3nM).
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using Hiseq PE Cluster Kit (Illumina) according to the manufacturer’s instructions. After cluster generation, the DNA libraries were sequenced on Illumina Hiseq platform and 150 bp paired-end reads were generated.

The Oriental nymphalid butterfly genus Symbrenthia Hübner, 1819 (Nymphalinae), commonly called jesters, belongs to the tribe Nymphalini which has 14 recognized species (Fric et al., 2004 (link), 2022 (link); Kunte, 2010 ), mainly distributed in the Oriental region and reaching New Guinea and the eastern Palaearctic (Bozano & Floriani, 2012 ). Symbrenthia lilaea Hewitson, 1864, the common jester, is distributed primarily in India, China, Nepal, Bhutan, Myanmar, and Pakistan (Mehra et al., 2018 ). An adult female of S. lilaea was collected from Debregeasia sp. (Urticaceae) on July 7, 2019 at Kuankuoshui National Nature Reserve (28°17′25″N, 107°12′09″E; collected by Zhanglan) in Zunyi, Guizhou Province, China (Figure 1). A single leg of S. lilaea was stored in 100% ethanol at 20°C and sent to Guangzhou Ruike Gene Technology Co. for mitogenome extraction and sequencing. For Illumina sequencing, genomic DNA was isolated using TIANamp Genomic DNA Kit (Tiangen). The Illumina sequencing library was generated using Truseq Nano DNA HT Sample Prep Kit (Illumina). The complete mitogenome was sequenced using high‐throughput sequencing on the Illumina Novaseq 6000 platform with an average insert size of 350 bp and a paired‐end 150 bp (PE 150) sequencing strategy to generate a sequencing data not less than 2 GB. Raw reads were trimmed of adapters using Trimmomatic (Bolger et al., 2014 (link)).

Each amplicon was extracted with the illustra GFX PCR DNA and Gel Band Purification Kit (GE, UK). The amplicon mixtures were pooled with equal quantities of DNA, then a total amount of 150 ng of amplicons was used as input material for the DNA library preparations. The sequencing library was generated using the Truseq Nano DNA HT Sample Prep Kit (Illumina, USA) following the manufacturer’s recommendations, and index codes were added to each sample. DNA fragments were ligated with the adapter for Illumina sequencing, followed by further PCR amplification. Then the PCR products were purified (SPRIselect reagent, Beckman) and DNA size spectra were determined using an Agilent 2100 Bioanalyzer and quantified with a Qubit fluorometer (Invitrogen, Carlsbad, California, USA.). Finally, the DNA libraries were sequenced using the Illumina Miseq platform, and 300 bp paired-end reads were generated. Sequencing output was deposited in GenBank (Supplementary Table 1).

Whole‐genome sequencing was performed on DNA from tumor and matched blood samples. The mean tumor purity was estimated to be greater than 90%. A sequencing library was constructed using a Truseq Nano DNA HT Sample Prep Kit (FC‐121‐4003, Illumina) and sequenced on the Illumina HiSeq X platform to an average depth of 50× for tumor samples and 30× for matched blood samples, with 99% coverage of the known genome. DNA sequencing and integrative analysis of the data in this study were completed by Novogene Bioinformatics Institute. To identify the biallelic mutation, the PCR product was gel purified and cloned into the pGEM^® T vector (Promega). Plasmids were isolated from single colonies for the identification of POU6F2 mutations and DNA sequencing.