The Illumina Mate Pair Library Preparation kit was used for library construction following the manufacturer's instructions and two samples were run on one lane of an Illumina HiSeq2000 with 50bp reads. The sequences were aligned to hg19 using BWA and a BAM file containing only the clustered discordant reads with clustered mates was created. Breakpoints with more then five discordant reads were identified, as described previously (4 (link)).
Mate pair library preparation kit
Manufactured by Illumina
Sourced in United States
The Mate Pair Library Preparation Kit is a laboratory tool designed for the construction of mate pair libraries. It enables the preparation of DNA libraries for sequencing applications, allowing the analysis of genomic structural variations and rearrangements.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (2 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Hiseq2000 flow cell lanes, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Truseq dna sample preparation kit, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Nextera mate pair library preparation kit, by Illumina (1 mentions)
Truseq dna library preparation kit, by Illumina (1 mentions)
Hiseq 4000 genome analyzer, by Illumina (1 mentions)
Bcl2fastq program, by Illumina (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
7 protocols using mate pair library preparation kit
1
Mate Pair Library Sequencing for Structural Variants
2
Chloroplast Genome Assembly from Illumina Sequencing
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Total DNA was extracted from young leaves with the DNeasy Plant Mini Kit (Qiagen, CA, USA). DNA was sheared by nebulization with compressed nitrogen gas, yielding fragments of 300 bp in length, and fragmentation quality was checked on a Bioanalyzer 2100 (Agilent Technologies). Paired-end libraries were prepared with the Mate Pair Library Preparation Kit (Illumina, San Diego, California, USA) in accordance with the manufacturer’s instructions. Genomic DNA was sequenced on a single lane with multiplexing on HiSeq2000 flow cell lanes (Illumina Inc.).
For each species, the raw reads were assembled into non-redundant contigs with Velvet1.2.07 [18 (link)], a de novo sequence assembly software package, with k = 30 and scaffolding contigs having a minimum length of 100 bp. All contigs were then mapped against the reference cp genome in Camellia sinensis [7 (link)] with BLAST (http://blast.ncbi.nlm.nih.gov/ ) similarity searches against the NCBI nr database by using the default search parameters. To identify the chloroplast contigs, all the returned contigs were blasted to the reference genomes. Primer walking and additional Sanger sequencing were then used to fill the gaps between the seven to twelve large contigs and to verify the junctions between the single-copy and the IRs regions (S1 Table ).
For each species, the raw reads were assembled into non-redundant contigs with Velvet1.2.07 [18 (link)], a de novo sequence assembly software package, with k = 30 and scaffolding contigs having a minimum length of 100 bp. All contigs were then mapped against the reference cp genome in Camellia sinensis [7 (link)] with BLAST (
3
Gorilla Male and Female Genome Sequencing
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To test DiscoverY on non-human data, gorilla male genomic DNA (ID KB3781) was extracted from a fibroblast cell line provided by the San Diego Zoological Society. An Illumina paired-end library was constructed using the TruSeq DNA Sample Preparation Kit. Two Illumina mate pair libraries were constructed from male genomic DNA (applying a narrow 7–8 kb and a broad 5–10 kb BluePippin DNA size selection) using Nextera Mate Pair Library Preparation Kit. All libraries were sequenced (2 × 151 bp) on the HiSeq2500 (Rapid mode) and concatenated together to provide a gorilla male dataset at ~20x depth of coverage for male coverage calculation. Gorilla female reads at low depth of coverage (7x) were also generated from an unrelated female. Gorilla female genomic DNA (ID 2000–0150) was isolated from liver provided by the Smithsonian Institution with the DNeasy Blood and Tissue kit (Qiagen). An Illumina paired-end library was constructed using the TruSeq DNA Sample Preparation Kit, and the library was sequenced (2 × 151 bp) on the HiSeq 2500 (Rapid mode).
4
Comprehensive Genomic Library Preparation
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Paired-end (170, 500, and 800 bp) and mate-pair (2 kb) genomic libraries were prepared using a TruSeq DNA Library Preparation kit (Illumina) and a Mate Pair Library Preparation kit (Illumina), respectively. Mate-pair libraries with larger insert sizes were constructed using a Nextera Mate Pair Library Preparation kit (Illumina). Ten micrograms of genomic DNA were tagmented in a 400 μl reaction and fractionated using SageELF, with the recovery of 11 fractions with 3–16+ kb. Each fraction was circularized and fragmented with a Covaris S2. Biotin-containing fragments were purified using Dynabeads M-280 streptavidin beads. Sequencing adapters (KAPA TruSeq Adapter kit) were attached using a KAPA Hyper Prep kit. The libraries were amplified for 10–13 cycles and purified with 0.8× AMpure XP. DNA libraries were then sequenced (~388× coverage) using Illumina HiSeq2000 (TruSeq libraries) and HiSeq2500 (Nextera libraries) at the Functional Genomics Center Zurich (FGCZ), University of Zurich, Switzerland (Supplementary Table 1 ).
5
Illumina Mate Pair Sequencing for Structural Variants
6
Genomic DNA Isolation from Lushui Leaves
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Fresh leaves were collected from Lushui, Yunnan province in South China and were identified based on morphology. Total genomic DNA was isolated from fresh leaves using the DNeasy Plant MiniKit (Qiagen, CA, USA). DNA and voucher specimens of sampled species were deposited in the herbarium of Chinese Academy of Inspection and Quarantine. DNA was sheared by nebulization with compressed nitrogen gas, yielding fragments of 500 bp in length. Paired-end libraries were prepared with the Mate Pair Library Preparation Kit (Illumina, San Diego, California, USA) in accordance with the manufacturer’s instructions. Whole genome sequences were executed using Illumina Hiseq 4000 Genome Analyzer.
7
Mate Pair Library Sequencing for Structural Variants
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Up to 1 µg of high-quality genomic DNA was used for library generation with the Mate Pair Library Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The insert size length obtained varied from 2 to 4 kb. Mate-pair library was paired-end sequenced (2×100 bp) on Illumina HiSeq1500. Raw sequence reads were converted to the fastq format using the Illumina bcl2fastq program. After the quality control step, including adapter trimming and low-quality reads removal, reads were mapped to the reference genome (hg19) using bowtie2. 15 (link) Each read from read pair was mapped independently and only unique mapped reads were used for further analysis. The .bam file was used for narrowing down the region where the translocations were located. A custommade package for the programming language 'R' was applied for detection of discordant reads (paired reads where one read of the pair maps to one chromosome and the second read maps to another chromosome). Only clusters of 15 or more similar discordant reads between chromosomes with the translocation were further analysed. For visual verification of the results Integrative Genomics Viewer (IGV) was used. 16
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