Libraries were constructed by shearing genomic DNA and ligating Illumina paired-end adaptors. The constructed DNA libraries were hybridized to Agilent Human All Exon Target Enrichment kit V1, which was designed to capture 165,637 coding exons and their flanking regions (37.8 million bases, 71.6% in exons with average length of 228 bp). The purified capture products were then amplified and subjected to 72 base paired-end sequencing on the Illumina GAII instrument according to Illumina's standard protocol.
Paired end adaptor
Manufactured by Illumina
Sourced in United States
Paired-end adaptors are DNA adaptor sequences used in next-generation sequencing workflows. They attach to the ends of DNA fragments, enabling the sequencing of both ends of the fragments.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (7 mentions)
Ampure spri beads, by Beckman Coulter (6 mentions)
S220 sonicator, by Covaris (6 mentions)
Quant it picogreen, by Thermo Fisher Scientific (3 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Pcr ampli cation, by Bio-Rad (3 mentions)
Sureselect human all exon v6 dna kit, by Agilent Technologies (3 mentions)
Gaii instrument, by Illumina (2 mentions)
Human all exon target enrichment kit v1, by Agilent Technologies (2 mentions)
Hiseq 2000 sequencer, by Illumina (2 mentions)
Biotin 14 datp, by Thermo Fisher Scientific (2 mentions)
Pe pcr 1, by Illumina (2 mentions)
Pe pcr 2.0 primers, by Illumina (2 mentions)
Agilent sureselect human all exon v6 kit, by Agilent Technologies (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Klenow fragment, by New England Biolabs (2 mentions)
T4 dna polymerase, by New England Biolabs (2 mentions)
34 protocols using paired end adaptor
1
Exome Sequencing Library Construction
2
mRNA Isolation and Sequencing
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mRNA was isolated from 5 μg total RNA using Dynabeads mRNA DIRECT (Invitrogen) and was fragmented with RNA fragmentation reagent (Ambion). First strand cDNA synthesis was done using the SuperScript III First-Strand Synthesis System and 3 μg μl−1 random hexamers (Invitrogen), followed by second strand synthesis with DNA Polymerase I and RNase H. After purification, a sequencing library was generated from the double-stranded cDNA by using paired-end adaptors (Illumina) and the NEBNext DNA Sample Prep Reagent Set 1 (NEB). This library was sequenced using an Illumina Genome HiSeq2000.
3
High-throughput Targeted NRY Sequencing
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Genomic DNA of the selected samples were sheared using Bioruptor UCD-200 (Diagenode, Liège, Belgium) to 200–250 bp length, then were fixed to blunt-end, added 3′-A tail, and ligated with barcode-linked Illumina paired-end adaptors (Table S1 ). Ligation products were amplified by PCR, and 300 – 350 bp sections were extracted through agarose gel electrophoresis. Except for one sample (YCH53), the others were pooled into 8 pools, with 10–15 samples in equal amount in each pool (Table S1 ). NRY was enriched using custom designed bait library (see below) of G3360-90000 SureSelect kit for Illumina paired-end (Agilent, Santa Clara, CA, US) (the baits were listed in Table S5 ). After another round of amplification, the pools went through single-end or paired-end sequencing with either GAIIx or HiSeq2000 sequencer for 100 or 2×100 cycles (Illumina, San Diego, CA, US).
4
Hi-C Library Generation Protocol
5
Chloroplast DNA Genome Sequencing Protocol
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Total genomic DNA was isolated from 100 mg of fresh leaves using a modified CTAB method. The DNA concentration (>50 ng µL−1) was measured using a NanoDrop spectrophotometer. The isolated DNA was fragmented into small pieces using sonication. After end reparation and A-tailing, the short DNA fragments were ligated with the Illumina paired-end adaptors. Based on gel electrophoresis, the suitable fragments were purified and selected as templates for next-step PCR amplification to create the final DNA library. The quality and quantity of the DNA library were measured using the Agilent 2100 Bioanalyzer. Finally, the library was sequenced from both the 5′ and 3′ ends using Illumina NovaSeq6000 PE150 Sequencing platform (Illumina, CA, USA). By use of NGSQCToolkit v2.3.3, the raw reads were filtered to remove the linker sequence and low-quality reads defined as having more than 10% bases with Q-value <20, and thus high-quality clean reads were obtained. The clean reads were then assembled using SPAdes (Bankevich et al., 2012 (link)) 3.10.1 (http://cab.spbu.ru/software/spades/ ) software with CP genome of F. cirrhosa as reference (NCBI accession number NC_024728.1). Finally, LSC/IR and SSC/IR junctions were further verified by Sanger sequencing.
6
Exome Sequencing of Patients and Offspring
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Genomic DNA of the patients and offspring was isolated from peripheral blood mononuclear cells. The protocol was approved by the Ethics Committee of Peking University People's Hospital, and informed consent was obtained from the subjects. The exome sequences were efficiently enriched from 1.0 μg genomic DNA using the Agilent SureSelect Human All Exon V6 kit (Agilent Technologies). Qualified DNA was randomly fragmented to an average size of 180~280 bp; then DNA fragment was end-repaired and phosphorylated, followed by A-tailing and ligation at the 3' ends with paired-end adaptors (Illumina). At last, DNA library was sequenced on a Hiseq 4000 (Illumina) for paired-end 150 bp reads. Valid sequencing data were mapped to the reference genome (UCSC hg19) by Burrows-Wheeler Aligner software (BWA) [9 (link)]. SAMtools [10 (link)] and Picard Tools (http://broadinstitute.github.io/picard/ ) were utilized to sort the results and mark duplicate reads, respectively. Single nucleotide variants (SNVs) and insertions and deletions (Indels) detected were annotated with the ANNOVAR software (http://annovar.openbioinformatics.org/en/latest/ ) [11 (link)].
7
Targeted Exome Sequencing of Thyroid Genes
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A Qiagen DNA extraction kit (Qiagen, Hilden, Germany) was used to extract DNA samples from blood clots using the column method. The following is the process of library preparation: (1) genomic DNA randomly fragmented to an average size of 180–280 bp; (2) gDNA fragments repaired and phosphorylated; (3) A-tailing and ligation at the 3′ends with paired-end adaptors(Illumina) with a singel “T” base overhang and purification using AMPure SPRI beads from Agencourt. DNA library was hybridized with the probe library labeled with biotin in liquid phase. The probe library was specifically combined with the exons of 16 candidate genes: PAX8 (NM_013952), NKX2-5 (NM_004387), GLIS3 (NM_001042413), FOXE1 (NM_004473), NKX2-1 (NM_003317), TSHR (NM_000369), THRB (NM_000461), DUOX2 (NM_014080), DUOXA2 (NM_207581), DUOXA1 (NM_001276265), DUOX1 (NM_175940), TG (NM_003235), TPO (NM_001206745), SLC5A5 (NM_000453), IYD (NM_001164695; NM_203395), and SLC26A4 (NM_000441). Then, the prepared library was sequenced on an Illumina Hiseq x for 150 bp paired-end reads.
8
Whole-Exome Sequencing Protocol for Rare Genetic Variants
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The DNA of case no. II:4 (the proband) was analyzed by WES at Novogene Biotechnology Inc., with a mean read depth of target regions of 100X. The sequenced samples were prepared according to the Illumina standard protocol (Illumina, Inc.). First, genomic DNA was randomly fragmented to an average size of 180–280 bp by a Covaris S220 sonicator (Covaris, Inc.). The remaining overhangs were converted into blunt ends via exonuclease polymerase activities. Subsequently, DNA fragments were end-repaired and phosphorylated, followed by A-tailing and ligation at the 3′ends with paired-end adaptors (Illumina, Inc.). DNA fragments with ligated adapter molecules on both ends were selectively enriched by PCR. After PCR amplification, libraries were hybridized with a liquid phase containing a biotin-labeled probe and magnetic beads with streptomycin were used to capture the exons of genes. Captured libraries were enriched by PCR to add index tags to prepare for sequencing. Products were purified using an AMPure XP system (Beckman Coulter, Inc.) and quantified using the Agilent high-sensitivity DNA assay on the Agilent Bioanalyzer 2100 system (Agilent Technologies, Inc.). Finally, the DNA library was sequenced on Illumina HiSeq4000 for paired-end 150 bp reads.
9
Exome Sequencing of Affected Children
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Whole exome sequencing was performed for the two affected children (III-1, III-2) at the Beijing Novogene Bioinformatics Technology Co., Ltd (Beijing, China). Exome sequences were enriched using an Agilent liquid capture system (Agilent SureSelect Human All Exon V6; Agilent Technologies, Santa Clara, CA, U.S.A.) according to the manufacturer’s protocol. First, genomic DNA was randomly fragmented to an average size of 180–280 bp using a Covaris S220 sonicator (Covaris, Brighton, U.K.). Second, the DNA fragments were end-repaired and phosphorylated, followed by A-tailing and ligation at the 3′ ends with paired-end adaptors (Illumina, San Diego, CA, U.S.A.) with a single ‘T’ base overhang, and purified using AMPure SPRI beads from Agencourt (Azincourt, France). Then, the size distribution and concentration of the libraries were determined using an Agilent 2100 Bioanalyzer and qualified by using real-time PCR. The DNA libraries were then sequenced on an IlluminaHiSeq 4000 sequencer for paired-end 150 bp reads at Beijing Novogene Bioinformatics Technology Co., Ltd. The raw data were saved as a FASTQ (fq) format file.
10
Exome Sequencing Library Construction
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Libraries were constructed by shearing genomic DNA and ligating Illumina paired-end adaptors. The constructed DNA libraries were hybridized to Agilent Human All Exon Target Enrichment kit V1, which was designed to capture 165,637 coding exons and their flanking regions (37.8 million bases, 71.6% in exons with average length of 228 bp). The purified capture products were then amplified and subjected to 72 base paired-end sequencing on the Illumina GAII instrument according to Illumina's standard protocol.
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