Snails were stored in 96% ethanol at −20°C before DNA extraction. For pools of Swedish localities SWn1, SWn2, and SWn4, we used DNA extractions from individual snails used by Ravinet et al. (37 (link)) and pooled them in equimolar amounts after Qubit fluorometer quantification. For the rest of pools, DNA was extracted for batches of five individuals by combining pieces of foot muscle tissue of 1-mm3 size from five snails in one tube. Extractions were done using modified CTAB protocol from (56 ). DNA quality and quantity were assessed by agarose gel electrophoresis and Qubit fluorometer and pooled in equal amount to produce the final pools for sequencing. These samples were further purified using a Zymo Genomic DNA Clean-up and Concentrator kit and subjected to quality and quantity control as above. Genomic libraries were built with the Illumina TruSeq PCR-Free Kit (Illumina, California, USA), insert sizes between 350 and 670 bp, and were sequenced in an Illumina HiSeq2500 machine with an SBS Kit v4 chemistry at SciLifeLab (Uppsala, Sweden).
Sbs kit v4 chemistry
Manufactured by Illumina
Sourced in Sweden
The SBS kit V4 chemistry is a core component of Illumina's sequencing technology. It enables DNA sequencing by synthesis, the fundamental process that powers Illumina's sequencing platforms. The kit provides the necessary reagents and consumables required to perform this essential step in the sequencing workflow.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500 sequencer, by Illumina (4 mentions)
Truseq dna pcr free ht kit, by Illumina (4 mentions)
S2 instrument, by Covaris (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Qubit fluorescence assay, by Thermo Fisher Scientific (3 mentions)
Paired end cluster kit v4, by Illumina (2 mentions)
Truseq pcr free kit, by Illumina (1 mentions)
Hiseq 2500 machine, by Illumina (1 mentions)
Cbot instrument, by Illumina (1 mentions)
5 protocols using sbs kit v4 chemistry
1
Pooled DNA Extraction and Sequencing of Snails
2
Whole Genome Sequencing of Blood Samples
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Genomic DNA from collected blood samples was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific, Waltham, MA, USA) and sheared with an S2 instrument (Covaris, Woburn, MA, USA). Library preparation was conducted using the TruSeq DNA PCR-Free HT Kit (Illumina, San Diego, CA, USA). Individual DNA libraries were measured using the 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) qPCR and Qubit (Thermo Fisher Scientific). All flow cells were sequenced on a HiSeq 2500 sequencer (Illumina) using the SBS kit V4 chemistry (Illumina). FastQC was used for quality control, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm. The identification of SNPs and indels and genotyping were performed across all samples simultaneously using standard hard-filtering parameters or variant quality score recalibration according to the GATK Best Practices recommendations. WGS was performed using a minimum median coverage of 30×.
3
Whole Genome Sequencing Protocol
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Whole blood was collected for genomic DNA extraction. Genomic DNA was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific) and sheared with an S2 instrument (Covaris). Library preparation was carried out using the TruSeq DNA PCR-Free HT Kit (Illumina). Individual DNA libraries were measured by 2100 Bioanalyzer (Agilent) qPCR and Qubit (Thermo Fisher Scientific). Normalized DNA libraries were combined into five-sample pools per flow cell in all eight lanes and clustered on a cBot instrument (Illumina) with Paired-End Cluster Kit V4 (Illumina). All flow cells were sequenced on the HiSeq2500 sequencer (Illumina) using the SBS Kit V4 chemistry (Illumina). FastQC was used to check read quality, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm (10 (link)). Single nucleotide variants (SNVs) and indel identification and genotyping were performed across all samples simultaneously using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations. WGS was presented with a minimum, median coverage of 30X.
4
Whole Genome Sequencing of Blood Samples
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Genomic DNA from collected blood samples was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific) and sheared with an S2 instrument (Covaris). Library preparation was carried out using the TruSeq DNA PCR-Free HT Kit (Illumina). Individual DNA libraries were measured with 2100 Bioanalyzer (Agilent) qPCR and Qubit (Thermo Fisher Scientific). All flow cells were sequenced on a HiSeq 2500 sequencer (Illumina) using SBS kit V4 chemistry (Illumina). FastQC was used to check read quality, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm [34 ]. The identification of SNPs and indels and genotyping were performed across all samples simultaneously using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations [35 (link)].WGS was performed with a minimum, median coverage of 30X.
5
Whole Genome Sequencing of Human Samples
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Whole blood was collected for genomic DNA extraction. Genomic DNA was quantified with a Qubit fluorescence assay (Thermo Fisher Scientific) and sheared with a S2 instrument (Covaris). Library preparation was carried out using the TruSeq DNA PCR-Free HT kit (Illumina). Individual DNA libraries were measured by 2100 Bioanalyzer (Agilent) qPCR and Qubit (Thermo Fisher Scientific). Normalized DNA libraries were combined into five-sample pools per flow cell in all eight lines and clustered on a cBot instrument (Illumina) with Paired-End Cluster Kit V4 (Illumina). All flow cells were sequenced on the HiSeq. 2500 sequencer (Illumina) using SBS kit V4 chemistry (Illumina). FastQC was used to check read quality, and the resulting reads were aligned to the hg19 reference genome with the BWA-MEM algorithm16 . SNV and indel identification and genotyping were performed across all samples simultaneously using standard hard filtering parameters or variant quality score recalibration according to GATK Best Practices recommendations17 (link),18 . WGS was performed with a minimum median coverage of 30X.
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