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老鼠模型WES研究文章一篇

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发表于 2017-3-1 10:07:05 | 显示全部楼层 |阅读模式
我非常想学习,可惜时间有限!
http://onlinelibrary.wiley.com/doi/10.1002/path.4517/full

Materials and methods
Generation of Brca2-null, Trp53-null mammary tumoursMice carrying the Blg–Cre transgene [25] were mated with animals with both loxP-flanked Brca2 and loxP-flanked Trp53 containing alleles [26] generating Blg–Cre Brca2f/f Trp53f/f animals [27]. Mammary tumour and spleen tissue from mutant animals were excised from humanely killed, tumour-bearing mice. Part of the tumour was fixed in 4% phosphate-buffered formalin overnight for histological analysis and immunohistochemistry and part was snap-frozen on dry ice for isolation of DNA.


Genomic DNA preparationGenomic DNA was isolated from tumours and spleens using the DNeasy Blood and Tissue kit (Qiagen, UK), according to the manufacturer's instructions. DNA was quantified using the Qubit 2.0 kit (Life Technologies, UK).


Whole-exome DNA sequencingGenomic DNA (3 µg) was fragmented to 200 bp using a Covaris E Series instrument (Covaris, MA, USA) and the resultant library subjected to DNA capture, using the 50 Mb SureSelect Mouse All Exon kit (Agilent, CA, USA), according to the manufacturer's instructions. Illumina paired-end libraries were prepared from the captured target regions and quantified using a Bioanalyzer DNA chip (Agilent), followed by sequencing on a HiSeq2000 platform (Illumina, San Diego, CA, USA), acquiring 2 × 76 bp reads. Casava software (v. 1.8, Illumina) was used to make base calls. Sequences were output in fastq format. Reads failing the Illumina chastity filter were removed before further analysis. The raw data from this sequencing procedure are now deposited on the European Nucleotide Archive (ENA), Accession No. PRJEB6674 (http://www.ebi.ac.uk/ena/data/view/PRJEB6674).


Read mapping and detection of somatic mutations in exome sequencingBurrows–Wheeler Alignment (BWA, v. 0.5.9) was used to align reads to the mouse reference genome (GRCm37) [28]. Duplicate sequence reads (PCR-derived duplicates) were removed from further analysis at this point. Base quality recalibration, realignment around indels and variant calling were performed using the Genome Analysis Tool Kit (GATK, v. 1.0-6144-gdd92a14), using the Broad best practice variant detection workflow [29]. The MuTect algorithm (v. 1.1.4) was also used to identify somatic single nucleotide mutations in targeted exons [30]. Small insertions and deletions detected in the tumour sample that were absent in the matched normal were considered to be candidate somatic mutations. Variants called in regions not covered by the exome capture probes and variants marked as low quality (QUAL < 20) were excluded. Candidate somatic mutations were also assessed to confirm their validity by visualizing sequencing data, using the Broad Integrative Genomics Viewer tool [31]. The Protein Variation Effect Analyzer (PROVEAN; v. 1.1.3) software tool was used to predict whether a mutation has an impact on the biological function of a protein.


Detection of copy number alterations in exome sequencingCopy number alterations were predicted using exome DNA sequencing data and the CONTRA (v. 2.0.4) and CoNIFER (v. 0.2.2) software packages, using default parameters[32, 33].


Validation sequencingTo validate somatic mutations by Sanger sequencing, PCR amplicons encompassing the candidate mutation sites were sequenced in tumour and spleen DNA, using standard methods. PCR primers for amplification and sequencing were designed using the UCSC In Silico PCR tool [34].


Comparison with human somatic mutation dataThe likely human orthologues of the mouse Brca2-null, Trp53-null-deficient mammary tumour somatic mutations were identified using the MGI curated sets of homology (from NCBI Homologene build67) [35]. Tumour DNA sequence data from non-familial human breast cancer and ovarian cancer was accessed via the TCGA data portal [36].


Fluorescence in situ hybridization (FISH)BAC clones mapping to the Met locus (6A2) were used as FISH probes: RP23-239E3 (mid-position 17462960), RP23-73G15 (mid-position 17571929) and RP23-444 N4 (mid-position 17647861). Additionally, three BAC clones from the 6G3 chromosomal region (close to the telomere) were used as copy number reference for mouse chromosome 6: RP23-27 F7 (mid-position 147281394), RP24-127P8 (mid-position 147421860) and RP23-396 J18 (mid-position 147597428). BACs were ordered from BACPAC Resources Center at the Children's Hospital, Oakland Research Institute (CHORI, Oakland, CA, USA; http://bacpac.chori.org). These were labelled by nick translation with Spectrum Orange-dUTP (for Met probes) and Spectrum Green-dUTP (for reference probes) (Vysis Inc., Downers Grove, IL, USA). Routine FISH protocol for formalin-fixed, paraffin-embedded tissues was used, with slight modifications described in Supplementary materials and methods (see supplementary material).







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 楼主| 发表于 2017-3-1 10:07:22 | 显示全部楼层
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 楼主| 发表于 2017-8-17 10:33:33 | 显示全部楼层
五个月过去了,没想到我现在居然有时间仔细研究这篇文章了,这世界真奇妙
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