The QIAseq Human Breast Cancer Panel (93 genes, DHS-001Z; Qiagen) and the GeneRead Human Comprehensive Cancer Panel (160 genes, NGHS-501X; Qiagen) were used for library construction according to the manufacturer’s instructions. The libraries were assessed using a QIAseq Library Quant Assay Kit (#QSTF-ILZ-R; Qiagen) and applied to a MiSeq sequencer (Illumina, San Diego, CA, USA). The Qiagen web portal (https://www.qiagen.com/us/shop/genes-and-pathways/data-analysis-center-overview-page/ ) was utilized for data analysis [16 (link)]. For alignment, GenomeBrowse (http://goldenhelix.com/products/GenomeBrowse/index.html ) was used, and GRCH37 was used as the human genome reference. A commercial bioinformatic analysis service (Mitsubishi Space Software Co. Ltd., Tokyo, Japan) was asked to interpret the GeneRead Human Comprehensive Cancer Panel results. CNV was calculated using the cloud analysis pipeline of the Qiagen web portal, and corrected for the percent tumor content. Four or more and one or fewer CNVs were regarded as significant.
Qiaseq library quant assay kit
Manufactured by Qiagen
Sourced in Germany, United States, Canada
The QIAseq Library Quant Assay Kit is a laboratory equipment product designed for quantifying nucleic acid libraries. It provides a reliable and sensitive method to measure the concentration of sequencing libraries prior to sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Miseq sequencer, by Illumina (6 mentions)
Qiaseq human breast cancer panel, by Qiagen (5 mentions)
Generead human comprehensive cancer panel, by Qiagen (5 mentions)
Nebnext ultra 2 non directional rna second strand synthesis module, by New England Biolabs (4 mentions)
Superscriptiv first strand cdna synthesis system, by Thermo Fisher Scientific (3 mentions)
Qiaxcel dna high resolution kit, by Qiagen (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Qubit dsdna hs assay kit with qubit 3.0 fluorimeter, by Thermo Fisher Scientific (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Ion torrent s5xl instrument, by Thermo Fisher Scientific (2 mentions)
Miseq platform, by Illumina (2 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Phix control v3 library, by Illumina (1 mentions)
Qiaseq 1 step amplicon library kit, by Qiagen (1 mentions)
Iseq100 cartridge, by Illumina (1 mentions)
Local run manager software, by Illumina (1 mentions)
Qubit 3.0 fluorimeter, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
High pure ffpet rna isolation kit, by Roche (1 mentions)
27 protocols using qiaseq library quant assay kit
1
Comprehensive Breast Cancer Gene Panel Analysis
2
Microsatellite Amplicon Sequencing Protocol
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PCR (20 ng of DNA) and LT-RPA (5 ng of DNA, 10.5 mM of Mg(OAc)2 and 40 min incubation at 32°C) amplicons of HT17, NR24, CAT25, BAT26, D2S123, D18S61, D12ATA63, REN and HPRTII microsatellites (shorter primers were used for REN and HPRTII and are indicated in Supplementary Table S1 ) obtained for each blood sample were purified using Beckman Coulter™ Agencourt AMPure XP beads (Thermo Fischer Scientific), quantified and pooled in equimolar ratio. 900 ng of each pool were used for the ligation of dual-indexed adapters using QIAseq 1-Step Amplicon Library Kit (Qiagen) and no supplemental PCR amplification of the libraries was required. Libraries were assessed for quality and quantity with a Fragment Analyzer (Agilent) and QIAseq™ Library Quant Assay Kit (Qiagen) respectively. 50 pM of the pooled libraries were deposited on an iSeq 100 cartridge (Illumina) together with 20% of 50 pM PhiX control v3 Library (Illumina). Amplicon sequencing was performed on an iSeq 100 using 151 cycles of paired-end sequencing. FastQ files were generated for each sample using Local Run Manager Software (Illumina) and the read counts and sequences of each microsatellite allele were obtained from the aligned reads using an in-house developed Python code.
3
Targeted Enrichment of Plasma cfDNA
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The kit was purchased from QIAgen (Hilden, Germany). Sequencing libraries were prepared according to the manufacturer’s instructions. Briefly, 15 ng of plasma cfDNA fragments were end-repaired, A-tailed, ligated with UMI-barcoded adaptors. The adaptor-ligated libraries were target enriched with PCR using a panel of loci specific primers (6 cycles). The target enriched libraries were further amplified for 21 cycles with PCR and were size selected for an average fragment size of 350 bp. The library profile was analyzed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and quantified using QuBit dsDNA HS Assay kit with QuBit 3.0 fluorimeter (Thermo Fisher Scientific, San Jose, CA) and qPCR with QIAseq Library Quant Assay Kit (QIAgen, Hilden, Germany).
4
Illumina NextSeq 550 Sequencing Protocol
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Sequencing was performed on Illumina NextSeq 550. For a single sequencing run, a 4-multiplexed library was created by pooling the libraries, quantified using QuBit dsDNA HS Assay kit with QuBit 3.0 fluorimeter (Thermo Fisher Scientific, San Jose, CA) and qPCR with QIAseq Library Quant Assay Kit (QIAgen, Hilden, Germany), at an equal molar ratio. The multiplexed library was denatured and sequenced with NextSeq High Output kit 2 ×150 cycles paired.
5
Targeted RNA Sequencing of FFPET Samples
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Specimens were assessed for tumour content based on the H&E sections and manually macrodissected for RNA extraction using the High Pure FFPET RNA isolation kit (Roche Life Science). Library preparation was performed using the QIAseq Targeted RNA Custom Panel (Qiagen) targeting 533 genes (Supplementary Table S1 ), as per the manufacturer’s guidelines. Briefly, 450 ng of RNA was used for complementary DNA (cDNA) synthesis, followed by a polymerase chain reaction (PCR) for molecular barcoding with the BC primers. The barcoded cDNA was purified and then incubated with the LA and RS2 primers, followed by a universal PCR for library amplification and sample indexing. The final library was quantified using the QIAseq™ Library Quant Assay Kit (Qiagen) and sequenced on an Illumina Nextseq 500. Genes for the custom panel were selected based on the differentially expressed genes previously identified between responders and non-responders to anti-PD-1-based therapies [15 (link)], as well as drug targets currently available in clinical trials for the treatment of solid tumours (NCT02608268, NCT02554812 and NCT02668770).
6
cfDNA Sequencing Library Preparation and Enrichment
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The kit was purchased from Roche (Mannheim, Germany). Sequencing libraries were prepared according to the manufacturer’s instructions with the following modifications: plasma cfDNA was extracted using cfDNA extraction kit (TIANGEN Biotech Co. Ltd., Beijing, China) instead of the Avenio ctDNA isolation kit included. Briefly, 15 ng of plasma cfDNA fragments were end-repaired, A-tailed, ligated with UMI-barcoded adaptors and amplified with PCR (12 cycles). The adaptor-ligated libraries were hybridized for 18 hr with biotinylated oligo DNA baits and enriched with streptavidin-conjugated magnetic beads. The target enriched libraries were further amplified for 15 cycles with PCR and were size-selected for an average fragment size of 350 bp. The library profile was analyzed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and quantified using QuBit dsDNA HS Assay kit with QuBit 3.0 fluorimeter (Thermo Fisher Scientific, San Jose, CA) and qPCR with QIAseq Library Quant Assay Kit (QIAgen, Hilden, Germany).
7
Metagenomic Sequencing of Viral Samples
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Virus stocks was sequenced using a generic metagenomics sequencing workflow as described previously21 (link) with some modifications. For reverse-transcribing RNA into cDNA, SuperScriptIV First-Strand cDNA Synthesis System (Invitrogen, Germany) and the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, Germany) were used, and library quantification was done with the QIAseq Library Quant Assay Kit (Qiagen, Germany). Libraries were sequenced using an Ion 530 chip and chemistry for 400 base pair reads on an Ion Torrent S5XL instrument (Thermo Fisher Scientific, Germany).
8
Metagenomics Sequencing of Total RNA
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Extracted total RNA was sequenced using a universal metagenomics sequencing workflow (18 (link), 34 (link)). An amount of 350 ng total RNA per sample was reverse-transcribed into cDNA using the SuperScript IV first-strand cDNA synthesis system (Invitrogen, Germany) and the NEBNext Ultra II nondirectional RNA second strand synthesis module (New England Biolabs, Germany). Afterwards, cDNA was processed to generate Ion Torrent compatible barcoded sequencing libraries as described previously (2 (link), 18 (link)). Libraries were quantified with the QIAseq Library Quant assay kit (Qiagen, Germany) and subsequently sequenced on an Ion Torrent S5XL instrument using Ion 530 chips and chemistry for 400-bp reads (Thermo Fisher Scientific, Germany).
9
Full Genome Sequencing of Archived Viruses
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All viruses taken from archived samples were subjected to full genome sequencing essentially as described before [44 (link)]. Briefly, RNA was automatically extracted on a KingFisher Flex platform (Thermo Fisher Scientific, Waltham, MA, USA) using the RNAdvance Tissue Kit (Beckmann Coulter, Brea, CA, USA). Double stranded cDNA was generated from 350 ng total RNA using the SuperScript IV First-Strand cDNA Synthesis System (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA). After conversion into cDNA, fragmentation was achieved by ultrasonication on a Covaris M220 (Covaris, Brighton, UK). Subsequently, Ion Torrent-specific sequencing libraries were generated using the GeneRead L Core Kit (Qiagen, Hilden, Germany) together with IonXpress barcode adaptors (Thermo Fisher Scientific). After quantification (QIAseq Library Quant Assay Kit, Qiagen) and quality control (2100 Bioanalyzer, High sensitivity DNA Kit, Agilent Technologies, Santa Clara, CA, USA) of the libraries, sequencing was performed on an Ion Torrent S5XL instrument utilizing Ion 530 chips and reagents according to the manufacturer’s instructions.
10
Paired-end Genomic Library Preparation for T. affinis
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Amongst the twelve samples of T. affinis collected, a select male specimen (sample no. 7) was used for generation of paired-end genomic libraries using TruSeq DNA PCR-Free library preparation kit (Illumina Inc., USA). About 1100 ng of the isolated DNA was used to generate paired-end genomic library of insert size 350 (2 × 150) base pairs. The DNA was fragmented using focused ultrasonicator (Covaris M220, USA) to a desired length as per the protocol recommended by the manufacturer. Following TruSeq DNA PCR-Free library preparation kit subsequent clean-up of the fragmented DNA, blunt-end creation and adapter ligation was performed [28 ]. The mean peak size of the generated library was checked using Fragment Analyzer AATI 5200 (Agilent, USA). QIAseq Library Quant Assay Kit (Qiagen N.V., Germany) was used to quantify the library and the selected library was normalized to 4 pico-moles before sequencing. The library was sequenced using NextSeq550 (Illumina Inc., USA) and at the end of the sequencing run, high-quality paired-end reads were obtained.
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