Bcl convert

Manufactured by Illumina

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Bcl-convert is a software tool developed by Illumina for the conversion of BCL (Base Call) files to other data formats, such as FASTQ. It serves as a utility to enable efficient data processing and analysis workflows for Illumina sequencing data.

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Lab products found in correlation

Nextseq 2000, by Illumina (5 mentions) Dna prep kit, by Illumina (4 mentions) Nextseq 550, by Illumina (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Stranded total rna prep ligation with ribo zero plus kit, by Illumina (2 mentions) Phage dna isolation kit, by Norgen Biotek (2 mentions) Nextseq 2000 platform, by Illumina (2 mentions) Rnase a, by Thermo Fisher Scientific (2 mentions) Dneasy blood and tissue kit, by Qiagen (2 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions) Sequencing adaptor, by Illumina (1 mentions) Rnase free dnase 1, by New England Biolabs (1 mentions) Synergy lx multi mode reader, by Agilent Technologies (1 mentions) Bcl2fastq, by Illumina (1 mentions) Novaseq 6000 system, by Illumina (1 mentions) Dragen dna pipeline, by Illumina (1 mentions) Dragen bio it platform, by Illumina (1 mentions) Trizol protocol, by Thermo Fisher Scientific (1 mentions) Stranded mrna prep, by Illumina (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)

Spelling variants of this product

Bcl-convert (v.3.9.3) (8 citations) BCL Convert Conversion Software (2 citations)

13 protocols using bcl convert

Bacterial Genome Sequencing Workflow

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DNA was extracted from
overnight cultures (15 mL) using the Qiagen QIAamp DNA mini kit according
to the manufacturer’s protocol. Whole genome sequencing was
performed by SeqCenter as follows. Sample libraries were prepared
using the Illumina DNA Prep kit and IDT 10 bp UDI indices and sequenced
on an Illumina NextSeq 2000, producing 2 × 151 bp reads. Demultiplexing,
quality control, and adapter trimming were performed with bcl-convert*
(v3.9.3) [*bcl-convert: a proprietary Illumina software for the conversion
of bcl files to basecalls]. FastQ files were uploaded to the Bacterial
and Viral Bioinformatics Resource Center. First, the parent strain
was annotated using the Genome Annotation tool. Next, the variation
analysis tool was used to identify genetic differences in the mutant
genomes. The identified mutations were confirmed via sanger sequencing.

Rutherford J.T., Avad K., Dureja C., Norseeda K., GC B., Wu C., Sun D., Hevener K.E, & Hurdle J.G. (2024). Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target. ACS Infectious Diseases, 10(5), 1612-1623.

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Illumina-based transcriptome profiling protocol

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The Illumina sequencing adaptors were ligated onto the fragments. Constructed libraries were sequenced (300 cycles) using Illumina NextSeq550 (Illumina Inc), according to the manufacturer’s instructions. The Binary Base Call (BCL) files were converted to FASTQ files using the Illumina BCL Convert (v3.7.5). Raw-seq reads quality was assessed using FastQC (v0.11.9)² (18 ). Adapters and low-quality bases (Q<20 in 4bp sliding window) were trimmed using fastp (v0.20.1) (19 (link)). Specifically, seven bases SMARTer adapter from both ends of the reads will be trimmed for plasma cfRNA only. Clean RNA-seq reads were then mapped to the human genome from the Genome Reference Consortium (GRCh38) using STAR aligner (v2.7.7a) with the 2-pass mode (20 (link)). Alignments were quantitated using HTSeq (v0.13.5) (21 (link)) overlapping with the annotations in GENCODE human release 35. The definition of the biotypes was referenced to GENCODE³ (22 ). Gene expression estimation in terms of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) and differential expression analysis was performed by the R (v4.0.5)/Bioconductor package DESeq2 (v1.30.1) (23 (link)). Reactome Pathway Database (24 (link)) annotation was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID v2021) (25 (link)).

Jin N., Kan C.M., Pei X.M., Cheung W.L., Ng S.S., Wong H.T., Cheng H.Y., Leung W.W., Wong Y.N., Tsang H.F., Chan A.K., Wong Y.K., Cho W.C., Chan J.K., Tai W.C., Chan T.F., Wong S.C., Yim A.K, & Yu A.C. (2023). Cell-free circulating tumor RNAs in plasma as the potential prognostic biomarkers in colorectal cancer. Frontiers in Oncology, 13, 1134445.

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RNA-Seq Analysis of C. difficile Transcriptome

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C. difficile strains were cultured on 70:30 sporulation agar as described above and harvested at H₁₂ into 6 ml of 1:1:2 ethanol:acetone:dH₂O solution and stored at −80°C. RNA was isolated and Dnase-I treated (Ambion). Samples were sent to Microbial Genomics Sequencing Center (MiGS; Pittsburgh, PA) where library preparation was performed using Illumina’s Stranded Total RNA prep Ligation with Ribo-Zero Plus kit and 10bp IDT for Illumina indices. Sequencing was done on a NextSeq200 giving 2×50bp reads. Demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v3.9.3; Illumina; see reference above). Using Geneious Prime v2022.2.2, the reads were mapped to the reference genome (630∆erm; NC_009089.1). The expression levels were calculated and then subsequently compared using DESeq2 (80 (link)).

Edwards A.N, & McBride S.M. (2023). The RgaS-RgaR two-component system promotes Clostridioides difficile sporulation through a small RNA and the Agr1 system. bioRxiv.

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Phage DNA Isolation and Sequencing Protocol

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Phage stock (1 ml) was transferred into four separate 15 ml conical tubes. In each tube, 20 units (10 µl for 2,000 units/ml stock solution) of RNase-free DNase 1 (New England Biolabs, Ipswich Massachusetts) was added and incubated at room temperature for 15 min, then a Norgen Phage DNA Isolation Kit (Thorold, ON, Canada) was used to isolate the DNA. The concentration of DNA was measured using the Synergy LX Multi-Mode Reader (BioTek Instruments, Inc., Winooski, Vermont) nanodrop. Phage DNA libraries were prepared using the Illumina DNA prep kit and IDT 10 bp indices, and were sequenced on an Illumina NextSeq 2000 platform (San Diego, CA), producing 2 × 151 bp reads. The total 2,743,288 read pairs were demultiplexed, quality controlled and adapter trimmed with the Illumina bcl-convert (v3.9.3). The genome was assembled with SPAdes (v3.13.0). The average fold coverage was 15,727. All sequencing and assembly procedures were performed by the Microbial Genome Sequencing Center (Pittsburgh, PA).

Mankovich A.G., Maciel K., Kavanaugh M., Kistler E., Muckle E, & Weingart C.L. (2023). Phage-antibiotic synergy reduces Burkholderia cenocepacia population. BMC Microbiology, 23, 2.

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Sequencing Data Processing and Alignment

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The sequencing data were demultiplexed using the Illumina software BCL Convert (https://emea.support.illumina.com/sequencing/sequencing_software/bcl-convert.html). Adaptor sequences were trimmed and overlapping reads were collapsed using AdapterRemoval (v2.2.4)^{53 (link)}. Single-end collapsed reads of at least 30 bp and paired-end reads were mapped to human reference genome build 37 using BWA (v0.7.17)^{54 (link)} with seeding disabled to allow for higher sensitivity. Paired- and single-end reads for each library and lane were merged, and duplicates were marked using Picard MarkDuplicates (v2.18.26; http://picard.sourceforge.net) with a pixel distance of 12,000. Read depth and coverage were determined using samtools (v1.10)^{55 (link)} with all sites used in the calculation (-a). Data were then merged to the sample level and duplicates were marked again.

Barrie W., Yang Y., Irving-Pease E.K., Attfield K.E., Scorrano G., Jensen L.T., Armen A.P., Dimopoulos E.A., Stern A., Refoyo-Martinez A., Pearson A., Ramsøe A., Gaunitz C., Demeter F., Jørkov M.L., Møller S.B., Springborg B., Klassen L., Hyldgård I.M., Wickmann N., Vinner L., Korneliussen T.S., Allentoft M.E., Sikora M., Kristiansen K., Rodriguez S., Nielsen R., Iversen A.K., Lawson D.J., Fugger L, & Willerslev E. (2024). Elevated genetic risk for multiple sclerosis emerged in steppe pastoralist populations. Nature, 625(7994), 321-328.

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Whole-Genome Sequencing of Clostridioides difficile

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C. difficile strains were grown overnight in and harvested from 10 ml BHIS. After storing cell pellets overnight at -80°C, genomic DNA was isolated using a modified Bust ‘N’ Grab protocol [26 (link),78 (link)]. Contaminating RNA was removed by incubation with RNase A (Ambion) for 1 h at 37°C. Library prep and Illumina sequencing were performed by SeqCenter (Pittsburgh, PA). Briefly, sample libraries were prepared using the Illumina DNA Prep Kit and IDT 10bp UDI indices, and sequenced on an Illumina NextSeq 2000, producing 2x151bp reads. Demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v3.9.3; Illumina; https://support-docs.illumina.com/SW/BCL_Convert/Content/SW/FrontPages/BCL_Convert.htm). Using Geneious Prime v2022.3, the resulting reads were paired and trimmed using the BBDuk plug-in and subsequently mapped to the reference genome (630Δerm; NC_009089.1). The Bowtie2 plug-in was used to search for the presence of SNPs and InDels under default settings with a minimum variant frequency set at 0.85. Genome sequence files were deposited to the NCBI Sequence Read Archive (SRA) BioProject PRJNA986905.

Edwards A.N, & McBride S.M. (2023). The RgaS-RgaR two-component system promotes Clostridioides difficile sporulation through a small RNA and the Agr1 system. PLOS Genetics, 19(10), e1010841.

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Transcriptional Profile of C. difficile Strains

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C. difficile strains were cultured on 70:30 sporulation agar as described above and harvested at H₁₂ into 6 ml of 1:1:2 ethanol:acetone:dH₂O solution and stored at -80°C. RNA was isolated and Dnase-I treated (Ambion). Samples were sent to Microbial Genomics Sequencing Center (MiGS; Pittsburgh, PA) where library preparation was performed using Illumina’s Stranded Total RNA prep Ligation with Ribo-Zero Plus kit and 10bp IDT for Illumina indices. Sequencing was done on a NextSeq200 giving 2x50bp reads. Demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v3.9.3; Illumina; see reference above). Using Geneious Prime v2022.2.2, the reads were mapped to the reference genome (630Δerm; NC_009089.1). The expression levels were calculated and then subsequently compared using DESeq2 [83 (link)]. DESeq2 utilizes the Wald test to calculate P values which are then adjusted using the Benjamini-Hochberg test [83 (link)]. RNA-seq raw sequence reads were deposited to the NCBI Sequence Read Archive (SRA) BioProject PRJNA986905.

Edwards A.N, & McBride S.M. (2023). The RgaS-RgaR two-component system promotes Clostridioides difficile sporulation through a small RNA and the Agr1 system. PLOS Genetics, 19(10), e1010841.

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RNA-seq Analysis of Astyanax mexicanus

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RNA-seq reads were demultiplexed into FASTQ format allowing up to one mismatch using Illumina bcl-convert (v3.10.5). Subsequently, the reads were aligned to Astyanax mexicanus reference genome from University of California at Santa Cruz (UCSC) using STAR (v2.7.3a) (Dobin et al., 2013 (link)). The gene model retrieved from Ensembl, release 102 was used to generate gene read counts. The transcript abundance ‘TPM’ (Transcript per Million) was quantified using RSEM (v1.3) (Li & Dewey, 2011 (link)). Differentially expressed genes were determined using R package edgeR (v3.38.4) (Robinson, McCarthy, & Smyth, 2010 (link)). Prior to differential expression analysis, low-expression genes were filtered out based on a cutoff of 0.5 CPM (Counts Per Million) in at least one library. The resulting p-values were adjusted with Benjamini-Hochberg method using R function p.adjust. Genes with an adjusted p-value < 0.05 and a fold change of 2 were considered as differentially expressed.
Gene functional enrichment analysis or gene ontology (GO) analysis was performed using a custom script built over R package ‘clusterProfiler’ (v4.4.4) (Wu et al., 2021 (link)). Astyanax mexicanus Gene-GO terms retrieved from Ensembl BioMart were used to identify over-represented GO terms in the differentially expressed genes compared to the background list of all genes.

Biswas T., Rajendran N., Hassan H., Zhao C, & Rohner N. (2023). 3D spheroid culturing of Astyanax mexicanus liver-derived cell lines recapitulates distinct transcriptomic and metabolic states of in vivo tissue environment. bioRxiv.

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Illumina Sequencing of Clonal Phage Isolates

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For clonal phage isolates, DNA was isolated from high-titer phage stocks using the Norgen Phage DNA isolation kit (cat. no. 46800) according to manufacturer’s instructions and sequenced at SeqCenter (seqcenter.com) using Illumina sequencing. Sample libraries were prepared using Nextera-based library preparation (38 (link)) using IDT for Illumina 10 bp indices. DNA from clones RB-020 through RB-028 were sequenced on an Illumina NextSeq 550 and Clone ET013 was sequenced on an Illumina NextSeq 2000, producing 2 × 151-bp reads. For mixed populations, DNA was isolated from the full community (bacteria plus phage) revived from frozen mixed communities grown overnight at 37°C. The full-community DNA was isolated using the Qiagen DNeasy blood and tissue kit (cat. no. 69504) and then sequenced at Felix Biotechnology on an Illumina NextSeq 550 platform using Nextera DNA Flex amplicon library preparation. Postsequencing, quality control measures were conducted by the sequencing provider for individual clones: for RB-020 through RB-028, the metrics from the sequencer were used to guarantee that the number of bases with a quality score of Q30 or higher meets or exceeds the total ordered. The data were demultiplexed, and adapters were removed using bcl2fastq (Illumina). For ET013, demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v3.9.3; Illumina).

Burmeister A.R., Tzintzun-Tapia E., Roush C., Mangal I., Barahman R., Bjornson R.D, & Turner P.E. (2023). Experimental Evolution of the TolC-Receptor Phage U136B Functionally Identifies a Tail Fiber Protein Involved in Adsorption through Strong Parallel Adaptation. Applied and Environmental Microbiology, 89(6), e00079-23.

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Paired-End Sequencing on NovaSeq 6000 with DRAGEN

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Following paired-end sequencing with 2 × 101 bp reads on the NovaSeq 6000 system (Illumina, San Diego, California, USA), demultiplexing of samples was performed using BCL Convert (Illumina), and raw sequencing data were processed using the DRAGEN DNA Pipeline on the Illumina DRAGEN Bio-IT Platform (Illumina) v3.9. After adapter trimming, sequencing reads were aligned to GRCh38/hg38. Duplicates and reads with a mapping quality < 30 were removed from analysis. A second bam file with 90–150 bp fragments only was generated for SCNA analysis. In all of the following analysis regions overlapping with ENCODE blacklist [24 (link)] and the UCSC gap track [25 ] were excluded.

Hallermayr A., Wohlfrom T., Steinke-Lange V., Benet-Pagès A., Scharf F., Heitzer E., Mansmann U., Haberl C., de Wit M., Vogelsang H., Rentsch M., Holinski-Feder E, & Pickl J.M. (2022). Somatic copy number alteration and fragmentation analysis in circulating tumor DNA for cancer screening and treatment monitoring in colorectal cancer patients. Journal of Hematology & Oncology, 15, 125.

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