An in-house bioinformatics pipeline was used to process tumor-only WES data. FASTQ data were generated using the bcl2fastq tool (Illumina, v2.18) and run through FASTQC for quality confirmation. Reads were mapped to the human hg19 reference genome using the Burrows-Wheeler alignment tool21 . The resulting bam files were processed using GATK best practice workflow22 . GATK HaplotypeCaller and Platypus23 (link) were used to call variants. Copy number data was inferred from WES data through use of the CNVKit algorithm24 (link), using a pool of normal Hapmap cell line specimens as references. The variants identified by WES were further annotated by the MoCha Oncogenic MOI Annotator (MOMA: https://github.com/FNL-MoCha/moma ), a sequencing platform agnostic tool used to annotate variants as Mutations of Interest (MOIs) or Variants of Unknown Significance (VuS). These classifications are based on data from annotating variants with Annovar25 (link) and mapping variants to OncoKB26 .
Bcl2fastq tool
Manufactured by Illumina
Sourced in United States
The bcl2fastq tool is a software application developed by Illumina that converts sequencing data from the Illumina Genome Analyzer and NovaSeq systems into FASTQ format. The core function of this tool is to demultiplex sequencing data and generate FASTQ files, which are the standard file format for storing DNA sequencing reads.
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Lab products found in correlation
Novaseq 6000, by Illumina (4 mentions)
Nextseq 500, by Illumina (2 mentions)
Qiasymphony rna kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Miseq platform, by Illumina (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Truseq pe cluster kit v4 cbot hs, by Illumina (1 mentions)
Whole exome sequencing, by Illumina (1 mentions)
Kapa rna hyperprep, by Roche (1 mentions)
Truseq stranded mrna library prep kit, by Illumina (1 mentions)
Kapa rna hyperprep kit, by Roche (1 mentions)
Qubit rna iq assay, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Influx cell sorter, by BD (1 mentions)
Dsp dna midi kit, by Qiagen (1 mentions)
Hiseq x sequencing system, by Illumina (1 mentions)
15 protocols using bcl2fastq tool
1
Comprehensive Tumor-Only Whole Exome Sequencing Pipeline
2
Transcriptome Profiling via mRNA-Seq
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Total RNA was extracted from cells or 30 mg of mouse tissue using the RNeasy minikit (Qiagen). Purified RNA was tested on an Agilent 2200 TapeStation using RNA screentape. mRNA-seq libraries were prepared using the TruSeq Stranded LT (Illumina kit). Samples were run on the Illumina NextSeq 500 using a High Output 75 cycle kit (twice for 36 cycles, paired-end reads, single index). Sample data were demultiplexed using the bcl2fastq tool (Illumina) before read quality was assessed using the FastQC algorithm, and then reads were trimmed appropriately using the TrimGalore! tool (Babraham Bioinformatics). Data were then aligned to an hg19 or mm9 reference genome (Ensembl), as appropriate, using the Tophat2 algorithm (Trapnell et al. 2010 (link); Kim et al. 2013 (link)). FPKM determination and differential expression analysis were performed using the Cufflink and Cuffdiff tools, respectively, from the Cufflinks suite. PCA analysis was performed using custom R scripts, then plotted using the Rgl package in R. Heat maps showing FPKM values of named gene subsets were produced using custom R scripts, with cohort means of Z-scores plotted.
RNA sequencing data were uploaded to GEO under accession number GSE132371.
RNA sequencing data were uploaded to GEO under accession number GSE132371.
3
Variant Identification from Illumina Sequencing
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To identify variants, we first converted and demultiplexed BCL basecall files generated by Illumina sequencing to fastq files using the bcl2 fastq tool according to the user guide (v2.15, Illumina). The fastq files were mapped to the GRCh37 build of the human genome reference using BWA-MEM (v0.7.15-r1140). Mapped reads in SAM format were then compressed to the BAM format by Samtools (v1.3.1). The base quality score in the BAM files was calculated and the insert/deletion (InDel) regions were realigned with the Genome Analytic Toolkit (version 3.6). VarDict was used to identify variants against the human genome reference, and the variants were annotated with SnpEff (version 4.2).
4
RNA-seq Bioinformatics Pipeline for Differential Gene Expression
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After preparation according to the manufacturer’s protocol, bioinformatic analysis was performed initially from converting and de-multiplexing BCL basecall files to Fastq files using the bcl2fastq tool (Illumina Inc., San Diego, CA, USA) following an Illumina user guide. Adapter sequences and low-quality bases were trimmed using Atropos [13 (link)]. The trimmed paired-end reads were mapped to the GRCh37 build of the human genome reference using STAR [14 (link)]. Mapped reads in SAM format were transferred to the compressed BAM format by SAMtools [15 (link)]. Duplicate reads in the BAM files were marked using the Picard utility [16 ]. Gene-specific read counting was performed using featureCounts [17 (link)] according to the GENCODE gene model [18 (link)]. For differential gene expression analysis, DESeq2 [19 (link)] was applied to the gene table of the raw read counts. The criteria of significance were the adjusted p-value (false discovery rate) being less than 0.005 and the log2 fold change being greater than and equal to 3 or less than and equal to −3 (Figure 1 ).
5
Illumina MiSeq Sequencing Data Processing
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Each sequencing run of the Illumina MiSeq platform produced a BCL file which was converted to FASTQ format (using Illumina’s bcl2fastq tool). Sequencing reads that failed the Illumina chastity filter were removed. The FASTQ file was demultiplexed into separate FASTQ files corresponding to each sample using the demuxFQ tool (https://github.com/gdbzork/demuxFQ ) with the default settings. The sample barcodes are provided in Supplementary Table S11 . Each sample’s FASTQ file was then processed through a custom pipeline which we describe below.
6
cDNA Library Preparation for Illumina Sequencing
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The preparation of cDNA library was carried out using a NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, United States) following the manufacturer’s recommendations at the Biomarker Biotechnology Corporation (Beijing, China). The detailed instructions for library construction were described in a previous study (Wang et al., 2021a (link)). The quality of all cDNA libraries was assessed using a Bioanalyzer 2100 system (Agilent Technologies, United States). Before transcriptome sequencing, a cBot Cluster Generation System with TruSeq PE Cluster Kit v4-cBot-HS (Illumia, United States) was employed to generate clusters. Finally, all constructed libraries were sequenced using the Illumina HiSeq2500 system, and paired-end reads were generated and converted to fastq format using bcl2fastq tool (Illumina, United States).
7
Whole Exome Sequencing for Genetic Variant Analysis
8
Genome-wide Chromatin Profiling Protocol
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FASTQ files were generated with Illumina’s Bcl2Fastq tool (v2.20) or original FASTQ files were downloaded from GEO and European Nucleotide Archive (ENA) databases (see Extended Data Table 1 for a list of accession IDs). Depending on the deposited source file format, paired-end or single-end reads were mapped to mm9 reference genome using bowtie2 (v 2.3.5.1)58 (link) and samtools (v 1.10)59 (link) deduplicated using Picard (v 2.20.4) (http://broadinstitute.github.io/picard/ ) MarkDuplicates with default settings and filtered for blacklisted regions. Bigwig files were generated from resulting deduplicated and filtered bam files using deepTools (v 3.1.0)60 (link), normalized to 1x Genome Coverage (Reads Per Genomic Content) using --normalizeUsing RPGC --effectiveGenomeSize 2150570000. In 1x Genome Coverage tracks, values below 1 represent a depletion below the genome average whereas values above 1 indicate fold-enrichment above genome average. Unless stated otherwise, downstream analysis was performed on the normalized bigWig files. Where available, we evaluated all experimental replicates (see Supplementary Fig. 5 ). For simplicity, figures show the first replicate.
9
RNA-Seq Library Preparation and Sequencing
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RNA was isolated from biopsy using the QIAsymphony RNA Kit (Qiagen, Hilden, Germany) for tissue and quantified on the Qubit. Between 50 and 100 ng of RNA was used as input for the library preparation with the Roche KAPA RNA HyperPrep and RiboErase (Human/Mouse/Rat) kits on an automated liquid handling platform (Beckman Coulter). RNA was fragmented (high temperature in the presence of magnesium) to a target length of 300 bp. Barcoded libraries were sequenced as pools on either a NextSeq 500 (V2.5 reagents) generating 2 × 75 base read pairs or on a NovaSeq 6000 generating 2 × 150 base read pairs using standard settings (Illumina, San Diego, CA, USA). BCL output from the sequencing platform was converted to FASTQ using Illumina’s bcl2fastq tool (versions 2.17 to 2.20) using default parameters. RNA-Seq data were aligned using STAR45 (link) to GRCh37 resulting in unsorted BAMs (Binary Alignment Maps) including chimeric reads as output. Gene and transcript counts were generated and used for subsequent fusion detection using Isofox (https://github.com/hartwigmedical/hmftools/tree/master/isofox ).
10
Transcriptome Analysis of Potato Cyst Nematode Resistance
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Total RNA was isolated from the root tissues of the PCN-resistant Kufri Swarna and PCN-susceptible Kufri Jyoti cultivars by using the TRIzol method. The integrity and quality of RNA were analyzed using a Qubit fluorometer. Equimolar concentrations of RNA from three biological replicates were pooled to construct an Illumina NextSeq PE library. Two 75 bp pair-end libraries for resistant Kufri Swarna and susceptible Kufri Jyoti were constructed by using the Illumina Truseq stranded mRNA Library Prep Kit following the manufacturer’s instructions. The libraries were sequenced on a NextSeq500 instrument with 2 × 75 chemistry. The resulting image files in the bcl format were converted to FASTQ with 2 × 75 bp reads using the bcl2fastq tool (Illumina, CA, USA). The raw read sequences were deposited in the Short Read Archive database at the U.S. National Center for Biotechnology Information (NCBI) (BioProject accession no. PRJNA488526).
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