RNA Sequencing - Building a FASTQ to BAM pipeline
Channel: Danny Arends
Date: 2022-10-23

Learn how to create a computational RNA sequencing pipeline using free and open source bioinformatics software. We will use the R to download the _Saccharomyces cerevisiae_ genome, transcriptome, and known SNPs from Ensembl and use a publicly available paired-end RNA-Seq data set from the Sequence Read Archive (SRA) to develop and test our pipeline in R. Learn how to trim reads, align them, as well as further steps like duplicate removal and howto use the GATK for base re-calibration. 

See the previous lecture here: https://youtu.be/PlqDQBl22DI
Code on GitHub: https://gist.github.com/DannyArends/04d87f5590090dfe0dc6b42e5e1bbe15
Presentation on OneDrive: https://1drv.ms/b/s!AtYWSYRMmSHZh4g7qrMcxofwNigzAg

Next time, we'll make the pipeline more generic, and use it to run several data sets and extract RPKM values from our aligned data for differential expression analysis.

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Chapters:
00:00:00 - Sound check and introduction
00:01:07 - Overview for today
00:02:53 - Install software I forgot
00:07:43 - Building a primary_assembly reference genome
00:22:25 - Download the transcriptome and known SNPs
00:27:42 - Creating the required genomic index files
00:34:38 - Building the RNA sequencing pipeline
00:37:00 - Execute external commands using R
00:42:42 - Static variables and the folder structure
00:47:00 - Automate downloading reads from SRA
00:51:27 - Trimming reads using Trimmomatic
01:05:43 - RNA paired-end alignment using STAR
01:12:00 - Samtools: BAM index and alignment statistics
01:28:12 - Picard tools: Duplicate removal and readgroup information
01:38:23 - GATK: Base re-calibration using known SNPs
01:45:24 - IGV: Visualize genome, transcriptome, and aligned reads
01:53:26 - What we'll do next time and Outro

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