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Pipeline publication

[…] b', 29 different HLOs were counted (n = 29). For cell quantification, 15 different HLOs were counted (n = 15). Statistical differences between groups were assessed with Prism software, using multiple t tests. All error bars represent SEM. Results were considered statistically significant at p < 0.05., Sequencing of HLOs (n = 3 D65, n = 3 D110) was performed by the University of Michigan DNA Sequencing Core, using the Illumina Hi-Seq platform. Sequencing of H9 Stem Cells (SC) and Definitive Endoderm (DE) was performed by the University of California, San Francisco DNA Sequencing Core using the Illumina Hi-Seq platform. All sequences were deposited in the EMBL-EBI ArrayExpress database using Annotare 2.0 and are catalogued under the accession number E-MTAB-3339 for the HLOs and E-MTAB-3158 for SC and DE. The University of Michigan Bioinformatics Core obtained the reads files and concatenated those into a single \xe2\x80\x98.fastq\xe2\x80\x99 file for each sample. The Bioinformatics Core also downloaded reads files from EBI-AE database (Adult lung Samples) and NCBI-GEO (SRA) database (Fetal lung samples) (). The quality of the raw reads data for each sample was evaluated using FastQC (version 0.10.1) to identify features of the data that may indicate quality problems (e.g., low quality scores, over-represented sequences, inappropriate GC content, etc). Initial QC report indicated over-representation of Illumina adapter sequences in samples from EBI-AE data set and NCBI-GEO data set. Adapter sequences were trimmed from the reads using Cutadapt (version 0.9.5) (). Briefly, reads were aligned to the reference transcriptome (UCSC hg19) using TopHat (version 2.0.9) and Bowtie (version 2.1.0.0) (). Cufflinks/CuffNorm (version 2.2.1) was used for expression quantitation and differential expression analysis (), using UCSC hg19.fa as the reference genome sequence and UCSC hg19.gtf as the reference transcriptome annotation. For this analysis, we used parameter settings: \xe2\x80\x98\xe2\x80\x93multi-read-correct\xe2\x80\x99 to adjust expression calculations for reads that map in more than one locus, as well as \xe2\x80\x98\xe2\x80\x93compatible-hits-norm\xe2\x80\x99 and \xe2\x80\x98\xe2\x80\x93upper-quartile \xe2\x80\x93norm\xe2\x80\x99 for normalization of expression values. Normalized FPKM tables were generated using the CuffNorm function found in Cufflinks. Transcriptional quantitation analysis in Cufflinks was conducted using the 64-bit Debian Linux stable version 7.8 (\xe2\x80\x98Wheezy\xe2\x80\x99) platform. The ' […]

Pipeline specifications

Software tools Annotare, FastQC, cutadapt, TopHat, Cufflinks