Computational protocol: Identification of non-coding genetic variants in samples from hypoxemic respiratory disease patients that affect the transcriptional response to hypoxia

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Protocol publication

[…] DNA was isolated from whole blood using Gentra Puregene Blood Kit (Qiagen, 158467) and regions of interest were polymerase chain reaction (PCR)-amplified using a 48.48 Fluidigm Access Array (Fluidigm Corporation, South San Francisco, USA). The resulting bar-coded amplicons were sequenced in a MiSeq apparatus (Illumina) under a 2 × 250 pair-ended format (Genomics Units, Science Park, Madrid). Reads were quality filtered according to the standard Illumina pipeline, de-multiplexed and fastq files were generated.Raw sequences generated by MiSeq (FASTQ format) were aligned to the February 2009 (GRCh37/hg19) assembly of the human genome with Bowtie2 (version 2.1.0) (,). Alignments were processed using Samtools (version 0.1.18) () and variants were called with bcftools filtering out those with mapping quality <30 or DP < 16. File manipulation and analysis were performed with custom scripts written in python (Python Software Foundation. Python Language Reference, version 3.4.3. Available at and R (R Core Team (2014). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL languages. We used Python version 3.4.3 and R version 3.2.2. For the visualization of aligned reads (BAM files) we used the Integrative Genomics Viewer, Integrated Genomic Viewer (IGV) (). For the treatment of genomic intervals we used the IRanges and Granges () libraries from Bioconductor version 3.2 ( The regions selected for binding along with the supporting experimental evidence can be interactively accessed at the UCSC genome browser: […]

Pipeline specifications

Software tools Bowtie2, SAMtools, bcftools, IGV, IRanges
Databases UCSC Genome Browser
Application Genome data visualization
Organisms Homo sapiens
Chemicals Oxygen