After the sequencing platform generated the sequencing images, the pixel-level raw data collection, image analysis, and base calling were performed by Illumina’s Real Time Analysis (RTA) software on a Dell PC attached to the HiSeq2000 sequencer. The base call files (*.BCL) were converted to qseq files by the Illumina’s BCL Converter and the qseq files were subsequently converted to FASTQ files for downstream analysis. The RNA-Seq reads from the FASTQ files were mapped to the mouse reference genome (NCBI37/mm10) and the splice junctions were identified by TopHat. The output files in BAM (Binary Alignment/Map) format were analyzed by Cufflinks to estimate the transcript abundance. The mRNA abundance was expressed in FPKM (fragments per kilobase of exon per million reads mapped).
Bcl converter
Manufactured by Illumina
The BCL Converter is a software tool that converts BCL (Base Call) files generated by Illumina sequencing instruments into standard data formats, such as FASTQ and BAM. It is a core component of the Illumina sequencing data analysis pipeline, enabling the transfer of raw sequencing data into formats compatible with downstream bioinformatics tools and workflows.
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6 protocols using bcl converter
1
RNA-Seq Data Processing Pipeline
2
Autism Spectrum Disorder Genetic Profiling
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Paired samples were obtained from the University of Maryland Brain and Tissue Bank as detailed in S1 Table . Individuals were diagnosed with ASD (n = 12) or were controls; criteria for diagnosing ASD included the Autism Diagnostic Interview-Revised (ADI-R), Childhood Autism Rating Scale (CARS), and Autism Diagnostic Observation Schedule (ADOS) as detailed S1 Table . DNA was extracted from tissue dissections according to protocols in the QIAGEN Genomic DNA Handbook. Exonic regions were selectively captured using Agilent SureSelectXT Human All Exon V5. Sequencing was performed at the Center for Inherited Disease Research at Johns Hopkins generating 100 bp sequence reads on an Illumina HiSeq. CIDRSeqSuite version 3.0.1 was used for processing of the raw data files. BCL files were converted to qseq format using Illumina’s BCL converter. qseq files were then demultiplexed and converted to FASTQ files using a custom demultiplexer. Paired-end alignment was performed using BWA aln to the 1000 genomes hg19/GRCh37 reference genome [34 (link)]. SAM files were sorted, converted to BAM, and duplicates were marked with Picard. GATK was used for local realignment and base quality score recalibration [35 (link),36 (link)]. Quality metrics for these data are provided in S2 Table .
3
Illumina RTA-Based Genome Alignment
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The Illumina Real Time Analysis (RTA) module performed image analysis, base calling, and the BCL Converter (CASAVA v1.8.2), generating FASTQ files containing the sequencing reads. These reads were aligned to the human genome (UCSC hg19 build) using the spliced-read mapper TopHat (v2.0.4) using the reference transcriptome to initially guide alignment, based on known transcripts of Ensembl Build GRCh37.67 (the “−G” argument in the software) 47 (link). The total number of aligned reads across the autosomal and sex chromosomes (dropping reads mapping to the mitochondria chromosome) per sample are provided in Table S1 .
4
Sequencing and Alignment of KSHV Genome
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After completion of Illumina sequencing, the raw output of BCL files were converted to the FASTQ format using the Illumina BCL Converter. Bowtie [38] , [86] (link), a short reads aligner application, was used to first align the sequencing tags against the KSHV genome (accession number NC_009333). The unaligned tags were subsequently aligned against human genome sequence hg19. For both alignments, Bowtie was run using default settings and additional options “–tryhard –best –strata” to obtain the alignments of the best quality. The resulting alignment SAM files were converted to various formats such as BAM and MPILEUP for downstream analysis. WIGGLE and TDF files were generated for visualization in the Integrative Genomics Viewer (IGV, Broad Institute). The ChIP data have been deposited in NCBI's Gene Expression Omnibus [87] (link) and are accessible through GEO Series accession number GSE52421 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52421 ).
5
Illumina RTA-Based Genome Alignment
6
RNA-Seq Analysis of Mouse Transcriptome
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Illumina's Real Time Analysis software was used for the pixel-level raw data collection, image analysis, and base calling. This software generated the base call files (*.BCL), which were converted to qseq files by the Illumina's BCL Converter. The qseq files were subsequently converted to FASTQ files for further downstream analysis. Using the mouse reference genome (NCBI37/mm9), the RNA-Seq reads from the FASTQ files were mapped to the mouse genome and splice junctions were identified by TopHat. The output files, which were in BAM (binary alignment/map) format, were analyzed by Cufflinks to estimate the transcript abundances. Cufflinks generated transcript structure predictions, which were then compared to the reference annotation Ensembl GTF version 65 by Cuffcompare. The mRNA abundances were expressed in FPKM (fragments per kilobase of exon per million reads mapped). RNA-Sequencing data was uploaded to the Gene Expression Omnibus database, accession number GSE58827.
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