Software installation

Set your python version to 2.7

module load python/2.7.11
python -V ## this should output 'Python 2.7.11'

Install anaconda

cd ~
wget https://repo.continuum.io/archive/Anaconda2-4.1.1-Linux-x86_64.sh
bash Anaconda2-4.1.1-Linux-x86_64.sh ## accept or confirm all prompts during installation

## re-logon to newton to activate anaconda
conda ## you should expect to see the help document for conda if you have installed anaconda successfully

conda config --add channels bioconda ## add the bioconda channel: a repository of ~1500 bioinformatic packages.

Install bioinformatic tools

conda install star -y
conda install hisat2 -y
conda install rampmap -y
conda install samtools -y
conda install skewer -y

Data preparation (from Arabidopsis thaliana)

Set up data directory

cd ~ 
mkdir RNASeq_lab_I
cd RNASeq_lab_I && mkdir 0_raw_data

Reference genome

cd ~/RNASeq_lab_I/0_raw_data
wget ftp://ftp.ensemblgenomes.org/pub/plants/release-28/fasta/arabidopsis_thaliana/dna/Arabidopsis_thaliana.TAIR10.28.dna.genome.fa.gz

Reference transcriptome

cd ~/RNASeq_lab_I/0_raw_data
wget ftp://ftp.ensemblgenomes.org/pub/plants/release-28/fasta/arabidopsis_thaliana/cdna/Arabidopsis_thaliana.TAIR10.28.cdna.all.fa.gz

Genome annotation

cd ~/RNASeq_lab_I/0_raw_data
wget ftp://ftp.ensemblgenomes.org/pub/release-28/plants/gtf/arabidopsis_thaliana/Arabidopsis_thaliana.TAIR10.28.gtf.gz
gunzip *gz ## uncompress all .gz files in your current directory

Raw reads (Experimental info)

cd ~/RNASeq_lab_I/0_raw_data
cp /data/home/mchen33/EPP622_2016_fall/rnaseq_labs_data.tar.gz ./
tar -xvzf rnaseq_labs_data.tar.gz 
mv ~/RNASeq_lab_I/0_raw_data/0_raw_reads/* ~/RNASeq_lab_I/0_raw_data/      ## move the reads files to '~/RNASeq_lab_I/0_raw_data/0_raw_data'
rmdir ~/RNASeq_lab_I/0_raw_data/0_raw_reads/      ## remove the empty directory '~/RNASeq_lab_I/0_raw_data/0_raw_data

RNASeq alignment/mapping: STAR, HISAT2 and RapMap

STAR

Index reference genome
```
cd ~/RNASeq_lab_I
mkdir alignment_STAR && cd alignment_STAR
mkdir genomeDir

STAR --runMode genomeGenerate     \
    --genomeDir ./genomeDir       \
    --genomeFastaFiles ../0_raw_data/Arabidopsis_thaliana.TAIR10.28.dna.genome.fa  \
    --runThreadN 4      \
    --sjdbGTFfile ../0_raw_data/Arabidopsis_thaliana.TAIR10.28.gtf   \
    --sjdbOverhang 101
```
- runMode genomeGenerate: run genome indices generation job, default is to run alignment.
- --genomeDir: specify the directory for storing genome indices
- --genomeFastaFiles: one or more FASTA files with genome reference sequences
- --runThreadN: the number of threads to use.
- sjdbGTFfile: The annotation file that STAR uses to build splice junctions database
- sjdbOverhang: specifies the length of genomic sequence around the annotated junction. Usually it is set to Readlength - 1.
- Command line to get the read length
  - Read 1: head -2 ../0_raw_data/DRR016140_1.1percent.fastq | awk "{print length}" | tail -2
  - Read 2: head -2 ../0_raw_data/DRR016140_2.1percent.fastq | awk "{print length}" | tail -2

Align the reads

We have 16 pairs of reads file. We can use a for loop to get the job done, instead of running the command 16 times.

cd ~/RNASeq_lab_I/alignment_STAR ## make sure you are in the right directory
mkdir alignment_output  ## create a directory to store the alignment output files

for i in `seq 25 40`
do
     STAR --genomeDir ./genomeDir       \
          --readFilesIn ../0_raw_data/DRR0161${i}_1.1percent.fastq ../0_raw_data/DRR0161${i}_2.1percent.fastq      \
          --outFileNamePrefix ./alignment_output/DRR0161${i}_  \
          --outSAMtype BAM SortedByCoordinate     \
          --runThreadN 4
done

--genomeDir: specifies the directory where you put your genome indices
--readFilesIn: your paired RNASeq reads files.
--outFileNamePrefix: your output file name prefix.
--outSAMtype: your output file type. Here we want the generated bam file to be sorted by coordination.
--runThreadN: the number of threads to be used.

HISAT2

Index reference genome

cd ~/RNASeq_lab_I
mkdir alignment_hisat2 && cd alignment_hisat2  ## create a directory for hisat2 alignment
mkdir genomeDir  ## again, we create a directory for the genome indices

hisat2-build -p 4 \
             ../0_raw_data/Arabidopsis_thaliana.TAIR10.28.dna.genome.fa \
             ./genomeDir/Athal_index

-p: specifies the number of threads to use
../0_raw_data/Arabidopsis_thaliana.TAIR10.28.dna.genome.fa: path to the reference genome
./genomeDir/Athal_index: the base of indices files that will be generated.

Align the reads

cd ~/RNASeq_lab_I/alignment_hisat2 ## go into the correct directory
mkdir alignment_output ## create a directory to store the alignment output files

for i in `seq 25 40`
do
     hisat2 ./genomeDir/Athal_index      \
            -1 ../0_raw_data/DRR0161${i}_1.1percent.fastq   \
            -2 ../0_raw_data/DRR0161${i}_2.1percent.fastq   \
            -S ./alignment_output/DRR0161${i}.sam             \
            -p 4
done

./genomeDir/Athal_index: path to the directory of genome indices
-1: specifies the first paired reads file
-2: specifies the second paired reads file
-S: output to a SAM file. Default is stdout.
-p: the number of threads to use.

Generate sorted bam files

hisat2 does not generate sorted bam file automatically. We will need to use samtools to convert the sam files to sorted bam fles.

cd ~/RNASeq_lab_I/alignment_hisat2
mkdir sorted_bam

for i in `seq 25 40`
do
   samtools sort ./alignment_output/DRR0161${i}.sam -o ./sorted_bam/DRR0161${i}_sorted.bam
done

RapMap: align reads to the transcriptome instead of the genome

Index reference genome

cd ~/RNASeq_lab_I
mkdir alignment_rapmap && cd alignment_rapmap

rapmap quasiindex   \
       -t ../0_raw_data/Arabidopsis_thaliana.TAIR10.28.cdna.all.fa \
       -i ./transcriptomeDir

quasiindex: builds a suffix array-based index
-t: specifies the path to the reference transcriptome file
i: specifies the location where the index should be written

Align the reads

cd ~/RNASeq_lab_I/alignment_rapmap
mkdir alignment_output

for i in `seq 25 40`
do
    rapmap quasimap \
           -i ./transcriptomeDir    \
           -1 ../0_raw_data/DRR0161${i}_1.1percent.fastq    \
           -2 ../0_raw_data/DRR0161${i}_2.1percent.fastq    \
           -t 4     \
           -o ./alignment_output/DRR0161${i}.sam    
done

quansimap: map reads using the suffix-array based method, should match the method you used for indexing.
-1: specifies the first set of reads from a paired library.
-2: specifies the second set of reads from a paired library.
-t: number of threads to use.
-o: path to the file where the output should be written.

Generate sorted bam files

RapMap does not generate sorted bam file automatically. We will need to use samtools to convert the sam files to sorted bam fles.

cd ~/RNASeq_lab_I/alignment_rapmap
mkdir sorted_bam

for i in `seq 25 40`
do
   samtools sort ./alignment_output/DRR0161${i}.sam -o ./sorted_bam/DRR0161${i}_sorted.bam
done

BAM files comparisons using `samtools flagstat` tool

STAR

cd ~/RNASeq_lab_I/alignment_STAR
mkdir flagstat_output

for i in `seq 25 40`
do
   samtools flagstat ./alignment_output/DRR0161${i}_Aligned.sortedByCoord.out.bam > ./flagstat_output/DRR0161${i}_flagstat.txt
done

hisat2

cd ~/RNASeq_lab_I/alignment_hisat2
mkdir flagstat_output

for i in `seq 25 40`
do
   samtools flagstat ./sorted_bam/DRR0161${i}_sorted.bam > ./flagstat_output/DRR0161${i}_flagstat.txt
done

RapMap

cd ~/RNASeq_lab_I/alignment_rapmap
mkdir flagstat_output

for i in `seq 25 40`
do
   samtools flagstat ./sorted_bam/DRR0161${i}_sorted.bam > ./flagstat_output/DRR0161${i}_flagstat.txt
done

Comparison of flagstat results

cd /data/home/mchen33/RNASeq_lab_I

cat alignment_STAR/flagstat_output/DRR016125_flagstat.txt
cat alignment_hisat2/flagstat_output/DRR016125_flagstat.txt
cat alignment_rapmap/flagstat_output/DRR016125_flagstat.txt

[Newton:sigma00 RNASeq_lab_I]$ cat alignment_STAR/flagstat_output/DRR016125_flagstat.txt
256344 + 0 in total (QC-passed reads + QC-failed reads)
16598 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
256344 + 0 mapped (100.00% : N/A)
239746 + 0 paired in sequencing
119873 + 0 read1
119873 + 0 read2
239746 + 0 properly paired (100.00% : N/A)
239746 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)



[Newton:sigma00 RNASeq_lab_I]$ cat alignment_hisat2/flagstat_output/DRR016125_flagstat.txt
259048 + 0 in total (QC-passed reads + QC-failed reads)
15780 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
255423 + 0 mapped (98.60% : N/A)
243268 + 0 paired in sequencing
121634 + 0 read1
121634 + 0 read2
234974 + 0 properly paired (96.59% : N/A)
237686 + 0 with itself and mate mapped
1957 + 0 singletons (0.80% : N/A)
96 + 0 with mate mapped to a different chr
25 + 0 with mate mapped to a different chr (mapQ>=5)



[Newton:sigma00 RNASeq_lab_I]$ cat alignment_rapmap/flagstat_output/DRR016125_flagstat.txt
335196 + 0 in total (QC-passed reads + QC-failed reads)
109792 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
331260 + 0 mapped (98.83% : N/A)
225404 + 0 paired in sequencing
112702 + 0 read1
112702 + 0 read2
220072 + 0 properly paired (97.63% : N/A)
220072 + 0 with itself and mate mapped
2666 + 0 singletons (1.18% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

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RNASeq_lab_I-alignment.md

RNASeq_lab_I-alignment.md

Software installation

Set your python version to 2.7

Install anaconda

Install bioinformatic tools

Data preparation (from Arabidopsis thaliana)

Set up data directory

Reference genome

Reference transcriptome

Genome annotation

Raw reads (Experimental info)

RNASeq alignment/mapping: STAR, HISAT2 and RapMap

STAR

HISAT2

RapMap: align reads to the transcriptome instead of the genome

BAM files comparisons using `samtools flagstat` tool

STAR

hisat2

RapMap

Comparison of flagstat results

Files

RNASeq_lab_I-alignment.md

Latest commit

History

RNASeq_lab_I-alignment.md

File metadata and controls

Software installation

Set your python version to 2.7

Install anaconda

Install bioinformatic tools

Data preparation (from Arabidopsis thaliana)

Set up data directory

Reference genome

Reference transcriptome

Genome annotation

Raw reads (Experimental info)

RNASeq alignment/mapping: STAR, HISAT2 and RapMap

STAR

HISAT2

RapMap: align reads to the transcriptome instead of the genome

BAM files comparisons using samtools flagstat tool

STAR

hisat2

RapMap

Comparison of flagstat results

BAM files comparisons using `samtools flagstat` tool