S Toolkit just before variant calling and integrated duplicate removal, neighborhood realignment about known indels and base top quality recalibration . The samples had been loaded individually towards the GATK UnifiedGenotyper application. Point mutations and expression information were plotted employing the Circos application . Comparison of point mutations was performed utilizing Venny. Accession numbers Binary sequence alignment/map files from complete exome sequencing information also as RNA-seq information were deposited in the database of the European Nucleotide Archive with accession number PRJEB4877 and are accessible by means of http://www.ebi.ac.uk/ ena/data/view/PRJEB4877. The sample accession numbers are ERS363578 and ERS363580 for complete exome sequencing information of LNCaP and C4-2B respectively. For the RNA-sequencing, the sample accession numbers are ERS363579 and ERS363581 for LNCaP and C4-2B cells respectively. Confirmation of non-synonymous variants Variants of interest had been confirmed by Sanger sequencing of amplified PCR merchandise. 17493865 Primers distinct to the region containing the variant to be tested were created working with the NCBI PrimerBlast and obtained from Integrated DNA Technologies. Polymerase chain reactions have been performed following regular protocols using Taq DNA polymerase. Amplification of specific PCR fragments was confirmed by agarose gel electrophoresis. Sanger sequencing was performed at LGC Genomics. Sequence trace files had been analyzed using Chromas Lite. RNA isolation LNCaP and C4-2B cells, with passage numbers of 30 and 43 respectively, were plated in 6-well plates and treated overnight with 1 nM R1881. The cells had been collected and washed with PBS. The cell pellet was utilised to extract total RNA applying the RNeasy Mini Kit from Qiagen. The high quality and purity of your RNA was inspected on a Nanodrop ND-1000 Spectrophotometer. The integrity with the RNA was verified on the BioAnalyzer in the Genomics Core of UZ Leuven. Final results Detecting point mutations with whole exome sequencing We performed a whole-exome re-sequencing study for each LNCaP and C4-2B cells utilizing one hundred base pair, paired-end reads around the Illumina platform. This generated 49 and 80 million reads for LNCaP and C4-2B respectively; for LNCaP cells, 74% in the exome was covered no less than 20x, versus 88% for C4-2B cells. RNA sequencing Following selection of polyA+ RNA, the RNA was converted into cDNA libraries making use of the TruSeq RNA Sample Preparation kit. Right after sequencing paired-end quick reads of 100 bp with all the HiSeq2000, normalized gene counts. The point mutations within the exomes had been detected working with the GATK pipeline to which additional filtering was applied: only mutations which had a minimum of 126 coverage and also a AKT inhibitor 2 site mutation frequency above 30% had been taken into account. Information were also filtered for absence of the base pair modify in dbSNP130. In addition, strand bias was eliminated manually. This resulted in lists of 2188 and 3840 non-synonymous point mutations in LNCaP and C4-2B cells, respectively. Only 1784 mutations were widespread (-)-Indolactam V amongst each cell lines, clearly indicating the accumulation of additional than 2000 26001275 extra mutations within the C4-2B genome. This huge difference in mutation load can’t be explained by the slightly lower coverage of the LNCaP exome. Most likely, these more C4-2B mutations have arisen during tumor progression and bone metastasis. Detecting point mutations in transcriptome sequencing Transcriptome sequencing was performed initially to determine differential gene expression. RNA was isolated from LNCaP and C4-2B ce.S Toolkit just before variant calling and included duplicate removal, local realignment around recognized indels and base excellent recalibration . The samples have been loaded individually to the GATK UnifiedGenotyper software program. Point mutations and expression information were plotted making use of the Circos computer software . Comparison of point mutations was performed making use of Venny. Accession numbers Binary sequence alignment/map files from whole exome sequencing information at the same time as RNA-seq information have been deposited inside the database of your European Nucleotide Archive with accession quantity PRJEB4877 and are accessible by way of http://www.ebi.ac.uk/ ena/data/view/PRJEB4877. The sample accession numbers are ERS363578 and ERS363580 for complete exome sequencing information of LNCaP and C4-2B respectively. For the RNA-sequencing, the sample accession numbers are ERS363579 and ERS363581 for LNCaP and C4-2B cells respectively. Confirmation of non-synonymous variants Variants of interest were confirmed by Sanger sequencing of amplified PCR solutions. 17493865 Primers precise for the region containing the variant to become tested had been developed using the NCBI PrimerBlast and obtained from Integrated DNA Technologies. Polymerase chain reactions have been performed following regular protocols using Taq DNA polymerase. Amplification of precise PCR fragments was confirmed by agarose gel electrophoresis. Sanger sequencing was performed at LGC Genomics. Sequence trace files were analyzed utilizing Chromas Lite. RNA isolation LNCaP and C4-2B cells, with passage numbers of 30 and 43 respectively, have been plated in 6-well plates and treated overnight with 1 nM R1881. The cells have been collected and washed with PBS. The cell pellet was applied to extract total RNA working with the RNeasy Mini Kit from Qiagen. The high quality and purity in the RNA was inspected on a Nanodrop ND-1000 Spectrophotometer. The integrity with the RNA was verified on the BioAnalyzer in the Genomics Core of UZ Leuven. Results Detecting point mutations with entire exome sequencing We performed a whole-exome re-sequencing study for each LNCaP and C4-2B cells utilizing one hundred base pair, paired-end reads around the Illumina platform. This generated 49 and 80 million reads for LNCaP and C4-2B respectively; for LNCaP cells, 74% with the exome was covered at least 20x, versus 88% for C4-2B cells. RNA sequencing After choice of polyA+ RNA, the RNA was converted into cDNA libraries utilizing the TruSeq RNA Sample Preparation kit. Right after sequencing paired-end brief reads of 100 bp with all the HiSeq2000, normalized gene counts. The point mutations in the exomes were detected making use of the GATK pipeline to which further filtering was applied: only mutations which had at the least 126 coverage and a mutation frequency above 30% have been taken into account. Data had been also filtered for absence from the base pair alter in dbSNP130. Additionally, strand bias was eliminated manually. This resulted in lists of 2188 and 3840 non-synonymous point mutations in LNCaP and C4-2B cells, respectively. Only 1784 mutations have been prevalent in between both cell lines, clearly indicating the accumulation of much more than 2000 26001275 extra mutations in the C4-2B genome. This large distinction in mutation load cannot be explained by the slightly reduced coverage of the LNCaP exome. Probably, these additional C4-2B mutations have arisen throughout tumor progression and bone metastasis. Detecting point mutations in transcriptome sequencing Transcriptome sequencing was performed initially to figure out differential gene expression. RNA was isolated from LNCaP and C4-2B ce.
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