Project description:Integration of Genomic and Transcriptional Features in Pancreatic Cancer Reveals Increased Cell Cycle Progression in Metastases - WGS mapped reads
Project description:UnlabelledWe describe a tool for quantifying the uniformity of mapped reads in high-throughput sequencing experiments. Our statistic directly measures the uniformity of both read position and fragment length, and we explain how to compute a P-value that can be used to quantify biases arising from experimental protocols and mapping procedures. Our method is useful for comparing different protocols in experiments such as RNA-Seq.Availability and implementationWe provide a freely available and open source python script that can be used to analyze raw read data or reads mapped to transcripts in BAM format at http://www.math.miami.edu/~vhower/ReadSpy.html.
Project description:We collected up to 6 separate analytes per patient from 14 HNSCC individuals and evaluated spatial (9 cases) and/or temporal (8 cases) variability using RNAseq. Sections taken from a frozen section using a crytostat were analysed for RNA quality was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies UK Ltd., Stockport, UK). Total RNA was converted into a library for sequencing on the HiSeq 2000 (Illumina Inc., San Diego, USA) using the TruSeq⢠stranded mRNA Sample Preparation Kit (Illumina Inc.). Briefly, poly-A mRNA was purified from total RNA (100ng) using the Poly(A) Purist Mag Kit (Life Technologies Ltd., Paisley, UK), according to the manufacturerâs instructions. The mRNA was then amplified and converted into cDNA, which was purified and used to construct libraries that were hybridized to the flow cell for single end (SE 35bp) sequencing. The protocol employed yielded 35-bp long reads. The quality of raw SE read data in FASTQ files were assessed and reads of low quality were trimmed or removed. SE reads were then mapped to the human genome (hg19) using TopHat (version 2.0.9) and, following the removal of multi-mapping reads, converted to gene specific read counts for annotated genes using HTSeq-count (version 0.5.4).