Truseq custom amplicon low input kit

(i) TS. TruSeq Custom Amplicon Low Input Kit (Illumina) and the targeted panel described above were used for library generation from reference strains and clinical samples, following the manufacturer's recommendations.
(ii) WGS. This was performed on reference strains only. WGS libraries were prepared using the Nextera XT system (Illumina), with 1.0 ng of input DNA, according to the manufacturer's recommendations. To obtain the in silico spoligotypes, the sequences obtained were used as input for the SpoTyping software (54 (link)); these results were used as the gold standard for linage classification.

Based on the presented results we aimed to focus on the analysis of both BRCA genes of interest and therefore used the TruSeq Custom Amplicon Low Input Kit (Illumina) in combination with a custom-designed BRCA gene panel. Selected FFPE samples were subjected to dual-pool amplicon-based library preparation. Subsequent sequencing of pooled libraries was performed on the MiniSeq sequencing platform using MiniSeq High Output Reagent Kit for 300-cycles.
Paired-end sequencing resulted in average 6115644.86 (6.1 Mio) paired-end passed filter reads per sample and mean amplicon coverage of 6774. Data analysis was conducted using on board Amplicon DS pipeline. Sequencing data was aligned to the reference genome UCSC hg19 using banded Smith–Waterman algorithm and variant calling was performed with Illumina Somatic variant caller. Filtering of all datasets was conducted manually according to predefined (custom) criteria.

High quality DNA was selected for NGS sequencing. We performed targeted panel sequencing to identify genetic variants within the UCP1 gene for MetS patients and controls. To build amplicon sequencing libraries for the coding region of the UCP1 gene, we used TruSeq Custom Amplicon Low Input Kit (Illumina, San Diego, CA, USA) with TruSeq Dual Index Sequencing Primers (Illumina, San Diego, CA, USA). For 3′UTR and 5′UTR regions of the UCP1 gene, we used AmpliSeq Illumina Custom DNA Panel (Illumina, San Diego, CA, USA) with AmpliSeq CD Indexes (Illumina, San Diego, CA, USA). The quality and quantity of the purified libraries was assessed by Qubit 3.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) using Qubit dsDNA HS Assay (ThermoFisher Scientific, Waltham, MA, USA) and TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) using High Sensitivity D1000 ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA). Only high-quality libraries were sequenced on the MiSeq System (Illumina, San Diego, CA, USA) using the V2 Illumina Sequencing Kit 500 (Illumina, San Diego, CA, USA).

A total of 100 ng input genomic DNA was used for library construction using Illumina TruSeq Custom Amplicon Low Input Kit (Illumina, San Diego, CA, USA). The libraries for the tumors, exposed-non-tumor (ENT) samples and the age-matched genomic controls were prepared following the manufacturer’s instructions, starting with hybridizing the oligo pool, extend and ligate the bound oligos, amplify the libraries with 28 PCR cycles. After library preparation, fragment analysis and quantification were performed with the TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) and Qubit 3.0 fluorimeter using the dsDNA HS assay kit (ThermoFisher Scientific, Waltham, MA, USA), respectively. Paired-end or single-end 150 base pair (bp) libraries for rat brain and cardiac tumors, interim sacrificed exposed-non-tumor (ENT) tissues, and age-matched unexposed and kidney control samples were sequenced on Illumina NextSeq (paired-end) and NovaSeq (single-end) platforms following manufacturer’s protocols (Illumina, San Diego, CA, USA).

Oligonucleotide primers were created in Illumina Design Studio and ordered through Illumina (Supplementary Data 1). Paired-end libraries for each sample were prepared using the TruSeq Custom Amplicon Low Input Kit (Illumina) according to the manufacturer’s instructions. This kit allows generation of up to 1536 amplicon targets over 96 samples. All DNA was diluted to 10 ng/μL during library preparation, or prepared with no dilution where concentrations were less than 10 ng/μL. An Agilent High Sensitivity D1000 ScreenTape System (Agilent Technologies) was used for measuring DNA concentration of prepared libraries. For sequencing, the MiSeq Reagent Kit v2 (300-cycles) (Illumina) reagents were used with the MiSeq sequencing platform.