We used the Illumina Experiment Manager to define the sample sheet, and the sample library was loaded into the MiSeq reagent cartridge for automated cluster generation and sequencing. The library quality control (QC) report was generated on the instrument using MiSeq Reporter software. The Illumina somatic variant analysis program align the reads to human reference genome hg19 with banded Smith-Waterman algorithm, and then we used VarScan2 [29 (link)] for calling somatic variants by the paired tumor-stroma mode with default program parameters: min coverage: 8x for normal, 6x for tumor, somatic P-value was 0.05. We selected P < 5×10–5 as the threshold for highly reliable somatic mutation detection. The wANNOVAR web server [30 (link), 31 (link)] was used for variants annotation, and those variants observed in the 1000 Genome Project, dbSNP138, or ESP6500 data sets were excluded since they are likely to be germline variants. Additionally, synonymous variants were removed since they are unlikely to change protein function. Detailed data filtering procedure is described in Fig. 1 . Raw sequence data was submitted to sequence read archive (SRA), and the accession number is SRP045337.
Experiment manager
Manufactured by Illumina
Sourced in United States
Experiment Manager is a comprehensive software solution designed to streamline the management of experiments and data within a laboratory setting. It provides a centralized platform for researchers to plan, execute, and analyze their experiments efficiently. The core function of Experiment Manager is to enable the organization, documentation, and integration of experimental workflows, facilitating seamless data collection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2fastq software, by Illumina (2 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Abi prism 3730xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Miseq system, by Illumina (1 mentions)
Seqscape software, by Thermo Fisher Scientific (1 mentions)
Nextgene software, by SoftGenetics (1 mentions)
Casava software, by Illumina (1 mentions)
Basespace, by Illumina (1 mentions)
8 protocols using experiment manager
1
Somatic Variant Calling Pipeline
2
Bisulfite Sequencing of Genomic DNA
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Bisulphite treatment of genomic DNA was performed with the EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer’s protocol. Amplicons were generated using region-specific primers (final concentration 10 μM each) with the recommended adaptors (Additional file 6 ). Purified PCR products were pooled in an equimolar ratio and sequenced after cluster formation on a MiSeq instrument benchtop sequencer with the sequencing-by-synthesis technology [16 (link)] according to the manufacturer’s protocol. Runs were set for “Generate FASTQ only” workflow in Illumina Experiment Manager. ESC and iPSC were analyzed as described elsewhere [33 (link)].
3
Comprehensive Genetic Profiling of Myeloid Malignancies
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Example 3
Using Sanger sequencing and NGS, mutations were analyzed in the following genes: ASXL1, ETV6, EZH2, IDH1, IDH2, NRAS, CBL, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and ZRSR2. Sanger sequencing was performed using a standard protocol. The primer pairs were designed to encompass >90% of reported mutations in these genes. Polymerase chain reaction (PCR) products were purified and sequenced in both forward and reverse directions using an ABI PRISM 3730XL Genetic Analyzer (Applied Biosystems, Foster City, Calif.). Sequencing data were base-called using sequencing analysis software and assembled and analyzed with SeqScape software (Applied Biosystems).
NGS was performed using the Illumina MiSeq system (San Diego, Calif.); NGS, amplification, and indexing were performed as recommended by the manufacturer. Amplicons were confirmed for each sample by running an agarose gel. Samples were pooled and the experiment sheet was generated using Illumina Experiment Manager. MiSeq Reporter was used for analysis and Variant Studio was used for calling. For confirmation of variant calling, NextGene software (SoftGenetics, State College, Pa.) was used. Average sequencing coverage across the entire coding regions was 4,000 in 94% of the sequenced amplicons. This reliably allowed detection of mutations if present in at least 3% of mutant DNA.
4
NextSeq500 Transcriptome Sequencing Protocol
5
16S rRNA Gene Sequencing for Microbiome Analysis
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Sequencing of the V3-V4 region of the 16S rRNA gene will be performed on all cases and control samples. We will aim to recruit no more that 50% of our cases from VRE positive patients. Preparation of 16S metagenomics libraries and deep sequencing will be carried out by the University of Calgary’s Core DNA Services. DNA libraries be prepared using the 16S Metagenomic sample preparation protocol (Illumina, San Diego, CA). The quality of the prepared library will be checked using Agilent TapeStation D1000 screen tape (Agilent Technologies, Santa Clara, CA), according to the manufacturer’s instructions.
Indexed DNA libraries are normalized to 4 nM and Illumina Experiment Manager used to build library plates and create sample sheet. Paired-end 300 bp sequencing will be performed on the MiSeq instrument using the V3 600 cycle MiSeq cartridge and MiSeq v3 reagents. The completed run will be demultiplexed with Illumina’s Casava software and stored in BaseSpace (Illumina) for downstream analysis.
Indexed DNA libraries are normalized to 4 nM and Illumina Experiment Manager used to build library plates and create sample sheet. Paired-end 300 bp sequencing will be performed on the MiSeq instrument using the V3 600 cycle MiSeq cartridge and MiSeq v3 reagents. The completed run will be demultiplexed with Illumina’s Casava software and stored in BaseSpace (Illumina) for downstream analysis.
6
Illumina BCL to FASTQ Conversion
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The sample sheet was prepared using the Illumina Experiment Manager (version 1.16). The BCL files were converted into FASTQ files using the bcl2fastq software (by Illumina, version 2.18.0.12). The option –no-lane-splitting was used to not split the FASTQ files by lane. All other default parameters were used.
7
SNP Calling for Potato Genomics
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The raw reads were preprocessed by simultaneously demultiplexing using Illumina Experiment Manager ver, 1.16.0. Raw Illumina sequencing reads are available at the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under Accession Number SUB14156832. The quality of the individual fastq files was assessed using FASTQC ver, 0.11.5. The SNP calling was conducted using the stacks software version 2. Briefly, reads were filtered using the process_radtags scripts to remove low quality reads with 0.15% of the length of reads. Then, the reads below 90% of base-call accuracy were discarded. Read depth ranged between 5 and 18 and depth of coverage was 5X.
The clean read were mapped to the most recent reference genome assembly of S.tuberosum using Bowtie2 ver. 2.3.2 [30 (link)]. The obtained Sequence Alignment Map files were converted to binary format and sorted with SAMtools 1.6. The loci were generated from the paired end data and SNP calling was performed using the STACKS software. Finally, population’s module of stacks was utilized to generate a variant call format (VCF) file. The reads were filtered to exclude variants with (minor allele frequency) MAF < 0.05, minGQ 15, using VCFtools. The PGDSpider 2.1.1.5 was utilized to convert the VCF file to other file format for downstream applications.
The clean read were mapped to the most recent reference genome assembly of S.tuberosum using Bowtie2 ver. 2.3.2 [30 (link)]. The obtained Sequence Alignment Map files were converted to binary format and sorted with SAMtools 1.6. The loci were generated from the paired end data and SNP calling was performed using the STACKS software. Finally, population’s module of stacks was utilized to generate a variant call format (VCF) file. The reads were filtered to exclude variants with (minor allele frequency) MAF < 0.05, minGQ 15, using VCFtools. The PGDSpider 2.1.1.5 was utilized to convert the VCF file to other file format for downstream applications.
8
Illumina Sequencing Data Processing
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The sample sheet was prepared using the Illumina Experiment Manager (version 1.16). The BCL les were converted into FASTQ les using the bcl2fastq software (by Illumina, version 2.18.0.12). The option -no-lane-splitting was used to not split the FASTQ les by lane. All other default parameters were used.
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