Primers were designed with NCBI Primer-BLAST tool with Tm range of 59–61 °C. The universal primer sequences (CS1 and CS2) were added at the 5′ end of the designed primers. All primer pairs were tested alone and in multiplexed PCR reactions using 10 ng of TaqMan® Control Human Genomic DNA (Thermo Fisher Scientific) in 10 μl reaction volumes. The coverage and performance of primers were analysed using 2200 TapeStation instrument (Agilent) and Hi-seq 4000. The primers were grouped together as 7-8plex, and primers in each group were chosen to target different genes in order to minimise non-specific amplification and cross-reactivity.
Hiseq 4000
Manufactured by Agilent Technologies
Sourced in Canada
The HiSeq 4000 is a high-throughput DNA sequencing system designed for large-scale genomic projects. It utilizes Illumina's sequencing-by-synthesis technology to generate high-quality sequencing data. The HiSeq 4000 is capable of producing up to 1.5 terabases of sequencing data per run, making it suitable for a wide range of applications, including whole-genome sequencing, transcriptome analysis, and targeted sequencing studies.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer, by Agilent Technologies (2 mentions)
Nextseq 500, by Illumina (2 mentions)
2200 tapestation instrument, by Agilent Technologies (1 mentions)
Truseq exome kit, by Illumina (1 mentions)
Novaseq 6000, by Agilent Technologies (1 mentions)
Bcl2fastq software, by Illumina (1 mentions)
Agilent tapestation, by Agilent Technologies (1 mentions)
Streptavidin beads, by Thermo Fisher Scientific (1 mentions)
Real time analysis software rta, by Illumina (1 mentions)
Nebnext ultra 2 directional kit, by New England Biolabs (1 mentions)
Truseq exome capture kit, by Illumina (1 mentions)
Sureselect human all exon v6 kit, by Agilent Technologies (1 mentions)
9 protocols using hiseq 4000
1
Multiplexed PCR Primer Design and Validation
2
Mouse Genome Sequencing and Variant Analysis
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Genomic DNA was sent to the company Genomics (New Taipei City, Taiwan) to establish DNA libraries. Sequencing was performed on Illumina HiSeq 4000, using Agilent Sure Select mouse, PE150, 5G data. The reads were aligned (bwa 0.7.17-r1188) to the mm10 mouse reference genome. Duplicated reads were marked by the PICARD (v2.17.2) and base quality recalibration was performed using GATK software (Genome Analysis Toolkit; Broad Institute; Cambridge, MA, USA). The filtered data were merged, re-aligned, and sorted to identify individual non-synonymous mutations.
3
Exome Sequencing and Variant Analysis
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Genomic DNA was extracted from peripheral blood leukocytes. Exomes were captured using either a TruSeq® Exome Kit (Illumina, San Diego, CA) on the NextSeq 500 system or SureSelect Human All Exons (Agilent Inc., Santa Clara, CA) on the Hiseq 4000 and Novaseq 6000 Systems. The reagents are according to the manufacturer's standard protocol. Sequences were aligned to the human reference genome (GRCh37) using the Burrows–Wheeler Aligner package version 0.7.15 [15 (link)]. Variant calling was performed using the Genome Analysis Tool Kit (GATK Best Practice V3.7; Broad Institute), which is called by HaplotypeCaller [16 (link)]. ANNOVAR was used to annotate genetic variants. The variant interpretation was limited to the 113 ACMG-recommended genes and G6PD. We included variants listed as PV and LPV according to VarSome's ACMG classification [17 (link)] with a final manual curation by the authors.
4
Single-cell RNA-seq of Mammary Epithelial Cells
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Following the same flow cytometry protocol as above, freshly sorted basal and luminal epithelial cells were submitted in a 1:1 ratio for sequencing using the manufacturer’s recommended Chromium (10× Genomics) and Illumina technologies. Two separate submissions of littermate-controlled, mammary epithelial cells were submitted to 10× Genomics. In each of the two submissions, two CO-treated MMECs were combined and two 4OHT-treated mice MMECs were combined and then were each submitted for sequencing. This resulted in a total of four CO-treated and four 4OHT-treated, 9-week-old mice used for downstream analysis. The amplified cDNA was constructed into 3′ expression libraries and pooled together in separate lanes of a 150-bp, paired-end chemistry flow cell. The Illumina HiSeq 4000 was used with an Agilent Bioanalyzer, and average library sizes ranged from 329 to 432 bp in length. The adapter sequence of the library was 124 bases in length. Basecalls were converted into FASTQ files by the University of Iowa Genomics Division using the Illumina bcl2fastq software. These each captured an estimated 3,546 to 5,545 cells per sample, which were submitted for sequencing with mean reads per cell ranging from 63,842 to 94,776.
5
Smart-Seq2 for Full-Length Transcriptomes
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RNA-Seq libraries were prepared from the picked cells following the Smart-Seq2 protocol for full length transcriptomes [104 (link)]. To minimize batch effects, primers, enzymes, and buffers were all used from the same lots for all libraries. Libraries were multiplexed, pooled, and purified using AMPure XP beads, quality was checked on an Agilent TapeStation, and libraries were sequenced as 51-bp single end reads on a HiSeq4000 at the UCSF Center for Advanced Technology.
6
Exome Sequencing and Variant Analysis
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Genomic DNA was isolated from a peripheral venous blood sample. We performed exome capture using the Agilent Sure Select v6 and massively parallel sequencing using the HiSeq 4000 platform as previously reported (Li and Durbin, 2009 (link); DePristo et al., 2011 (link)). Raw image analyses and base calling were performed using Illumina’s Pipeline with default parameters. Sequence data were aligned to the reference human genome using the Burrows–Wheeler Aligner (BWA) (Li and Durbin, 2009 (link)), and duplicate reads were removed using Picard tools. We used the Genome Analysis ToolKit (GATK) to perform the re-alignment and variation (SNP and InDel) detection (DePristo et al., 2011 (link)). Annovar was utilized to catalog the detected variations (Wang et al., 2010 (link)). Then, we filtered variations with a homo-polymer length >6 (and synonymous substitutions) or that were common (>2%) by dbSNP150 (http://www.ncbi.nlm.nih.gov/projects/SNP/ ), HapMap, the 1000 Genomes Project (http://www.1000 genomes.org ), Exome Aggregation Consortium (ExAC) database, and the Genome Aggregation Database (gnomAD, https://gnomad.broadinstitute.org ). Direct Sanger sequencing was performed for all patients and their parents to verify the genetic variants detected by WES. The data that support the findings of this study are available from the corresponding author upon reasonable request.
7
Exome-enriched RNA sequencing protocol
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Briefly, stranded RNAseq libraries were generated as per the manufacturer's recommendations, but without the transcriptome enrichment step (pre-capture libraries).
Transcriptome enrichment was achieved by the hybridization of the pre-capture library to the exome panels tested. Since the probe baits were biotinylated, hybridized libraries were captured using streptavidin beads (ThermoFisher, Waltham, MA) and PCR amplified-on-beads to generate a post-capture library. All post-capture libraries were subjected to quality control on an Agilent Bioanalyzer and normalized to 2nM. The postcapture libraries obtained from each capture platform were pooled, and each pool sequenced on one lane of a paired-end read flow cell for 2x100 cycles on a HiSeq4000 to obtain ~40M reads per sample. The primary processing of sequencing images was done using Illumina's Real Time Analysis software (RTA). CASAVA 1.8.2 software was then used to demultiplex samples and generate raw reads and respective quality scores (Supplementary Tables 1 and2). Libraries were made as described above using the NEBNext Ultra II Directional kit (PN-E7760, New England Biolabs, Ipswich, MA). 5µg of pooled indexed libraries (500ng each)
Transcriptome enrichment was achieved by the hybridization of the pre-capture library to the exome panels tested. Since the probe baits were biotinylated, hybridized libraries were captured using streptavidin beads (ThermoFisher, Waltham, MA) and PCR amplified-on-beads to generate a post-capture library. All post-capture libraries were subjected to quality control on an Agilent Bioanalyzer and normalized to 2nM. The postcapture libraries obtained from each capture platform were pooled, and each pool sequenced on one lane of a paired-end read flow cell for 2x100 cycles on a HiSeq4000 to obtain ~40M reads per sample. The primary processing of sequencing images was done using Illumina's Real Time Analysis software (RTA). CASAVA 1.8.2 software was then used to demultiplex samples and generate raw reads and respective quality scores (Supplementary Tables 1 and2). Libraries were made as described above using the NEBNext Ultra II Directional kit (PN-E7760, New England Biolabs, Ipswich, MA). 5µg of pooled indexed libraries (500ng each)
8
Whole Exome Sequencing of Tumor and Blood Samples
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WES was performed on two platforms. Thirty-three matched tumour/blood samples were sequenced on an Illumina Hiseq4000 using the Agilent sureselect V5 kit. Their overall tumour content was assessed using qpure54 (link) by comparing tumour SNP array data (2.5 M Illumina) with the matching normal blood. Eleven matched tumour/blood samples were sequenced on the Illumina NextSeq500 using the IDT pan-cancer spike in. Their cellularity was determined using the mean allele fraction. All samples contained > 20% tumour content. The mean tumour read depth was 439 × (range 254.78–1053.35) for the tumour samples and 216 × (range 52.98–1173.04) for the normal samples (Table S1 ).
9
Exome Sequencing Workflow: Capture, Align, and Variant Calling
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Exome capture was performed using either the TruSeq Exome Capture Kit (Illumina) or the SureSelect Human All Exon V6 Kit (Agilent). Sequencing was performed using the NextSeq500 (Illumina) or HiSeq4000 (Agilent) kit. A 75‐bp paired‐end run was performed. A mean raw coverage over 100‐fold was obtained for each sample. Sequence alignment to the human reference genome (GRCh37) was performed using the Burrows–Wheeler aligner (BWA), and variant calling was performed using the Genome Analysis Tool Kit (GATK V3.5, Broad Institute) (Wang, Li, & Hakonarson, 2010 ).
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