These are tutorials on a subset of tools available for processing raw RNAseq data. This is for two pipelines HISAT2_SAMtools_Stringtie_gffcompare_ballgown pipeline or HISAT2_SAMtools_Stringtie_PrepDEanalysis.py_DESeq2 pipeline. The first few steps are command line arguments and require installation of the following softwares. See Reference section for details on each software.

FASTQC HISAT2 SAMTOOLS Stringtie gffcompare

The description and tutorial of the tools was originally published here: https://www.nature.com/articles/nprot.2016.095 The sequenced samples used in tutorial was from original material Lappalainen et al. 2013 Nature and is accessible PMID:24037378. A useful portal to explore the dataset from original study is provided at https://www.ebi.ac.uk/Tools/geuvadis-das/. The dataset consists of lymphoblastic cell lines derived from 465 individuals. The individuals are either male or female and from one of five different populations: CEPH (CEU--Utah residents of Western and Northern European ancestry), Finns (FIN--Finland), British (GBR), Toscani (TSI--Italy) and Yoruba (YRI--Nigeria).

The sample data provided by JHU tools (HISAT2 and Stringtie) used for the analysis in tutorial(https://www.nature.com/articles/nprot.2016.095) is a subset of samples used in the original study. And the subset of samples (12) are further filtered for reads that map onto human chromosome X, for ease of analysis and reducing computational demand. So the index files provided by HISAT2 or Stringtie during installation(ex. chrX_tran.1.ht2...,which are used by HISAT2 for mapping) and reference annotated transcriptome provided by Stringtie during installation (ex. chrX.gtf, which is used by Stringtie for reference guided transcriptome assembly) are also of chromosome X(note: typically, homonid males have 1 X chromosome). For details of installing the bioinformatic tools and running the example dataset, please refer to Dave Tang's excellent tutorial. In this github project are presented options to expand the region covered, see below. And also a way to automate the pipeline (from aligning the reads, to sorting and conversion to bam file type, transcriptome assembly), a bash script is provided that can be modified.

**To get the complete set of samples with paired end RNA reads

• can ftp to the following website and perform a recursive download(limited by network capacity both locally and host's) wget -cr ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR188/

  **or individual samples for example: wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR188/ERR188021/ERR188021_1.fastq.gz

• At European Nucleotide Archive (ENA) search for ERP001942 https://www.ebi.ac.uk/ena/data/view/PRJEB3366 http://www.ebi.ac.uk/ena/data/view/ERR188021-ERR188482

• Or accessed at ArrayExpress with accession number E-GEUV-1 (There is also an miRNA and miRNA&RNA bundle) https://www.ebi.ac.uk/arrayexpress/

** To download the phenodata or metadata on samples visit: https://www.ebi.ac.uk/arrayexpress/experiments/E-GEUV-1/samples/?full=true

• or at command line curl -L "https://www.ebi.ac.uk/arrayexpress/files/E-GEUV-1/E-GEUV-1.sdrf.txt">geuvadis_complete_phenodata.txt **To process the downloaded datatable into a subset with just 3 columns of interest
• wc -l geuvadis_complete_phenodata.txt
• awk -F "\t" '{print $NF}' geuvadis_complete_phenodata.txt | wc -l
• awk -F "\t" '{print $34 " " $7 " " $36 " "}' geuvadis_complete_phenodata.txt >geuvadis_3columns.txt
• awk -F "\t" '{print $1 " " $2 " " $3}' geuvadis_3columns.txt
• sed -i 's/^Com.*$/ids " " sex " " population/' geuvadis_3columns.txt
• or sed -i 's/^Com.*$/ids \t sex \t population/' geuvadis_3columns.txt

To be consistent with Dave Tang's or JHU tutorial the relevant fields in the table for our purposes are "Characteristics[sex]"==gender of individual, "Characteristics[ancestry category] or Factor Value[ancestry category]"==geographic name of population, "Comment[ENA_RUN]"==sample names, "Comment[FASTQ_URI]"==sample file name and ftp path for download And thus you can construct a new data table with just the relevant information as described above using bash or Python and R below

** To generate the phenodata after downloading using Python or R

• python approach

  • import pandas as pd
  • geuvadis=pd.read_csv("geuvadis_complete_phenodata.txt", sep="\t", header=0)
  • geuvadis.shape
  • **The above should output: (924, 37)
  • geuvadis.iloc[0,:]
  • geuvadis.columns
  • geuvadis.iloc[0,33] sample name
  • geuvadis.iloc[0,35] The geo
  • geuvadis.iloc[0,6] gender

  **subset the dataframe/datatable to contain only relevant columns/variables

  • geuvadis_phenodata=geuvadis.iloc[:, [33,6,35]]

  **rename the column names to be consistent

  • geuvadis_phenodata.columns=['ids', 'sex', 'population']

**one can also further subset the phenodata to just two populations and abbreviate the geographic population label

* to write out the data frame as a csv file
* export_csv = df.to_csv (r'geuvadis_phenodata.csv', sep="," , index = None, header=True) 

• R approach

  • geuvadisdf<-read_csv("geuvadis_complete_phenodata.txt", sep="\t", header=T)
  • dim(geuvadisdf)
  • geuvadisdf<-geuvadisdf[,c(34,7,36]
  • names(geuvadisdf)<-c('ids', 'sex', 'population')
  • write.table(geuvadisdf, "geuvadisdf_phenodata.csv", sep=",")

** Another option(still computationally demanding) is to use the aligner/mapper: STAR

HISAT2 will use the reference genome for generating index files Although the reference genome is not used in this tutorial/analysis, it can be downloaded as follows curl -L "http://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz" > hg38.fa.gz

** However, and fortunately JHU has these whole genome chromosome indexes available for download

• wget -c ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/grch38.tar.gz

**Here is an example alignment command with HISAT2 using the JHU sample dataset(descibed above)

hisat2 -p 4 --dta -x chrX_data/indexes/chrX_tran -1 chrX_data/samples/ERR188428_chrX_1.fastq.gz -2 chrX_data/samples/ERR188428_chrX_2.fastq.gz -S ERR188428_chrX.sam

** [options]*

• -p <num_cores>: # of processor core threads
• --dta: downstream transcriptome assembly. Ex. for Stringtie etc. ** Main arguments
• -x:basename of the index for the reference genome, which excludes the ".1.ht2" etc.
• -1: comma-separated list of files containing mate 1s
• -2: comma-separated list of files containing mate 2s
• -S: file to write SAM alignments to. If not specified it is sent to stdout or console

Start at: https://genome.ucsc.edu/cgi-bin/hgTables and then choose as follows

• clade: Mammal
• genome: Human
• assembly: Dec. 2013 (GRCh38/hg38)
• group: Genes and Gene Predictions
• track: NCBI RefSeq
• region: genome (radio button)
• table: UCSC RefSeq(refGene)
• output format: GTF-gene transfer format(limited)
• output file: GRCh38_hg38_ucsc_annotated.gtf
• Send output to: radio buttons unchecked.
• file type returned: plain text

** Finally press "get output" button. ** Note that this reference genome was publicly available after the lymphoblastic cell line study was published

https://genome.ucsc.edu/cgi-bin/hgTables?hgsid=764817289_W7uU8Aj03hotPfShgOJGHr2RDr1M&clade=mammal&org=Human&db=hg38&hgta_group=genes&hgta_track=refS$

samtools sort -@ 4 -o ERR188428_chrX.bam ERR188428_chrX.sam **explanations of options arguments

• -@ 4: is the key value pair of CPU threads to use
• -o: is to specify the output filename
• last is the name of input file

** Once samples are mapped or aligned onto a reference genome, then Stringtie can be used to assemble the transcriptome. An example assembly command with Stringtie using the JHU sample dataset(descibed above)

stringtie map/ERR188428_chrX.bam -l ERR188428 -p 4 -e -G chrX_data/genes/chrX.gtf -o assembly/ERR188428_chrX.gtf

**If assembly for downstream analysis with ballgown then stringtie ERR188428_chrX.bam -l ERR188428 -p 4 -G chrX_data/genes/chrX.gtf -o assembly/ERR188428_chrX.gtf

explanations of options arguments

• path to input file
• -l:prefix for output transcripts
• -p: number of CPU threads to use
• -G: Reference annotation to guide assemble in either GTF/GFF3 files
• -o: output file path and name
• -e: this option is needed for DESEQ2. read bundles with no match to reference will be skipped http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual#deseq

**and subsequently to merge the transcripts (requires a mergelist text file) ls -1 assembly/* | cat > mergelist.txt **To generate a merge list text file stringtie --merge -p 4 -G chrX_data/genes/chrX.gtf -o stringtie_merged.gtf chrX_data/mergelist.txt

gffcompare -r chrX_data/genes/chrX.gtf -G -o merged stringtie_merged.gtf

[ -d ballgown ] || mkdir ballgown stringtie -e -B -p 8 -G stringtie_merged.gtf -o ballgown/ERR188044/ERR188044_chrX.gtf map/ERR188044_chrX.bam

** For convenience in using the binaries it is desirable, after installation, to link them to /usr/local/bin(this varies with OS you are using) so that they can be invoked without specifing their full path. ln -s /<full>/<path>/<to>/<file> /usr/local/bin

https://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc Downloaded FastQC v0.11.8 (Win/Linux zip file) unzip fastqc_v0.11.8.zip **make binary executable chmod 755 fastq ** To launch GUI ./fastqc ** To link it to /usr/local/bin ln -s ~/Desktop/FastQC/fastqc /usr/local/bin

wget -c ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/downloads/hisat2-2.1.0-Linux_x86_64.zip unzip hisat2-2.1.0-Linux_x86_64.zip **To link it to /usr/local/bin ln -s ~/Desktop/HISAT2/hisat2-2.1.0/* /usr/local/bin

wget https://github.com/samtools/samtools/releases/download/1.9/samtools-1.9.tar.bz2 tar -tvf samtools-1.9.tar.bz2 cd samtools-1.9.tar.bz2 ./configure **another option is ./configure --prefix=/where/to/install make ** it took 40seconds sudo make install ** To uninstall if needed sudo make uninstall ** to check the install steps make -n install **Linking to /usr/local/bin is unnecessary as the installation procedure moves the necessary binaries to appropriate folder

wget http://ccb.jhu.edu/software/stringtie/dl/stringtie-2.0.1.Linux_x86_64.tar.gz tar -xzvf stringtie-2.0.1.Linux_x86_64.tar.gz cd stringtie-2.0.1.Linux_x86_64 ln -s ~/Desktop/stringtie-2.0.1.Linux_x86_64/stringtie /usr/local/bin

git clone https://github.com/gpertea/gclib git clone https://github.com/gpertea/gffcompare cd gffcompare make release ln -s ~/Desktop/gffcompare/* /usr/local/bin ** may want to remove the unnecessary links later or initially select specific files to link instead of whole folder

**Alternatively, a precompiled version can be downloaded wget http://ccb.jhu.edu/software/stringtie/dl/gffcompare-0.11.4.Linux_x86_64.tar.gz tar xzvf gffcompare-0.11.4.Linux_x86_64.tar.gz cd gffcompare-0.11.4.Linux_x86_64

wget http://ccb.jhu.edu/software/stringtie/dl/prepDE.py chmod 755 prepDE.py **usage ** first generate a sample_lst.txt that contains sample name and its path (relative or absolute) at each line for ex. ERR188044 assembly/ERR188044_chrX.gtf * * * **Then run the script with text file containing the sample name and its corresponding path python2 prepDE.py -i sample_lst.txt

• Lappalainen T et al "Transcriptome and genome sequencing uncovers functional variation in humans". Nature(501):506–511 (2013.(http://dx.doi.org/10.1038/nature12531)
• Pertea M et al "Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown". Nature Protocols (11):1650–1667 (2016). https://www.nature.com/articles/nprot.2016.095
• https://www.ebi.ac.uk/Tools/geuvadis-das/
• https://www.internationalgenome.org/
• https://en.wikipedia.org/wiki/1000_Genomes_Project
• CEPH https://www.coriell.org/1/NIGMS/Collections/CEPH-Resources. https://en.wikipedia.org/wiki/Fondation_Jean_Dausset-CEPH
• Dave Tang's tutorial blog https://davetang.org/muse/2017/10/25/getting-started-hisat-stringtie-ballgown/
• FASTQC (version: 0.11.8) https://www.bioinformatics.babraham.ac.uk/projects/download.html#fastqc
• HISAT2(version: 2.1.0) https://ccb.jhu.edu/software/hisat2/index.shtml
• Stringtie (version: 2.0.1) http://ccb.jhu.edu/software/stringtie/index.shtml
• Gffcompare (version: ) http://ccb.jhu.edu/software/stringtie/gff.shtml#gffcompare or https://github.com/gpertea/gffcompare