RNASeqRData : RNASeqR vignette sample data explanation

Author: Kuan-Hao Chao (ntueeb05howard@gmail.com)

Contents

1 Introduction

RNASeqRData is a helper package for vignette in RNASeqR software package. This vignette shows the criteria of input_files and extraction process of mini example data.

2 input_files criteria

input.path.prefix is the parameter that stores the directory location of ‘input_files/’. Users have to prepare an ‘input_file/’ before running RNASeqR package workflow. The criteria of ‘input_file/’ are listed below:

• genome.name.fa: reference genome in FASTA file formation.

• genome.name.gtf: gene annotation in GTF file formation.

• raw_fastq.gz/: directory storing FASTQ files.

  • Support paired-end reads files only.

  • Names of paired-end FASTQ files : ’sample.pattern_1.fastq.gz’ and ’sample.pattern_2.fastq.gz’. sample.pattern must be distinct for each sample.

• phenodata.csv: information about RNA-Seq experiment design.

  • First column : Distinct ids for each sample. Value of each sample of this column must match sample.pattern in FASTQ files in ‘raw_fastq.gz/’. Column names must be ids.

  • Second column : independent variable for the RNA-Seq experiment. Value of each sample of this column can only be parameter case.group and control.group. Column name is parameter independent.variable.

• indices/ : directory storing HT2 indices files for HISAT2 alignment tool.

  • This directory is optional. HT2 indices files corresponding to target reference genome can be installed at HISAT2 official website. Providing HT2 files can accelerate the subsequent steps. It is highly advised to install HT2 files.

  • If HT2 index files are not provided, ‘input_files/indices/’ directory should be deleted.

3 Sample definition

The data in this experiment data package is originated from NCBI’s Sequence Read Archive for the entries SRR3396381, SRR3396382, SRR3396384, SRR3396385, SRR3396386, and SRR3396387. These samples were from Saccharomyces cerevisiae. To create mini data for demonstration purpose, reads aligned to the region from 0 to 100000 at chromosome XV were extracted. More details steps will be explained in the next chapter. Reference genome and gene annotation files, Saccharomyces_cerevisiae_XV_Ensembl.fa and Saccharomyces_cerevisiae_XV_Ensembl.gtf, are downloaded from iGenomes, Ensembl, R64-1-1.

4 Sample data preparation process

• fastq.gz files: fastq.gz files are aligned and analyzed in advanced in order to reduce the size of large raw fastq.gz, which are about 800M, and to keep the most differential expressed genes as far as possible. Reads aligned to the region from 0 to 100000 at chromosome XV were extracted only. Therefore, the size of these fastq.gz would be reduced to only about 5M. The following are the data processing steps:

1. SAMtools builds bam indexes of BAM files :

• ex: samtools index SRR3396381.bam

2. SAMtools extracts reads in certain range :

• ex: samtools view -b SRR3396381.bam "XV:0-100000" > SRR3396381.extracted.bam

3. SAMtools sorts extracted BAM files :

• ex: samtools sort -n SRR3396381.extracted.bam -o SRR3396381.sorted.bam

4. SAMtools gets splited fastq files :

• ex: bedtools bamtofastq -i SRR3396381.sorted.bam -fq SRR3396381_XV_1.fastq -fq2 SRR3396381_XV_2.fastq

5. gzip fastq files :

• ex: gzip SRR3396381_XV_1.fastq

Finally, mini data in this RNASeqRData package are created.

• Saccharomyces_cerevisiae_XV_Ensembl.fa: Only XV chromosome sequence are extracted.

• Saccharomyces_cerevisiae_XV_Ensembl.gtf: The whole Saccharomyces cerevisiae gtf annotation files.

5 Session Information

sessionInfo()

## R version 3.5.0 (2018-04-23)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 17.10
## 
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.7.1
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.7.1
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
## [1] grid      stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
## [1] png_0.1-7       BiocStyle_2.9.6
## 
## loaded via a namespace (and not attached):
##  [1] Rcpp_0.12.18       bookdown_0.7       digest_0.6.17     
##  [4] rprojroot_1.3-2    backports_1.1.2    magrittr_1.5      
##  [7] evaluate_0.11      stringi_1.2.4      rmarkdown_1.10    
## [10] tools_3.5.0        stringr_1.3.1      xfun_0.3          
## [13] yaml_2.2.0         compiler_3.5.0     BiocManager_1.30.2
## [16] htmltools_0.3.6    knitr_1.20