transcriptome assembly with Scythe-Sickle-Hisat-Stringtie-Ballgrown

gistfile1.txt

###IMPROVING FASTQ (note: apply the same to all your fastq files) ###

#deleting illumina adapters by https://github.com/vsbuffalo/scythe
./sickle se -f /home/uploader/flies/assembly4/3_cleaned.fastq -t sanger /home/uploader/flies/assembly4/3.fastq

#triming fastq by https://github.com/najoshi/sickle
sickle se -f /home/uploader/flies/assembly4/3_cleaned.fastq -t sanger -o /home/uploader/flies/assembly4/3.fastq -q 25

### Hisat http://ccb.jhu.edu/software/hisat/manual.shtml ###

#building hisat index
hisat-build dmel.fasta dmel.hisat

#extracting know splice sites to ease alignment for hisat
python extract_splice_sites.py in_gtf_filename > out_splice_site_filename

#aligning reads
hisat -x dmel.hisat -U 3.fastq -S 3_hisat.sam --known-splicesite-infile splicesites.txt

#12299676 reads; of these:
#  12299676 (100.00%) were unpaired; of these:
#    1665253 (13.54%) aligned 0 times
#    8570171 (69.68%) aligned exactly 1 time
#    2064252 (16.78%) aligned >1 times
#86.46% overall alignment rate

### StringTie ###

#convertion to bam by samtools http://www.htslib.org
samtools view -S -b 3_hisat.sam > 3.bam

#sorting
samtools sort 3.bam 3_sorted

#GTF creation

#generating gtf-s as well as coverage that will be required for further diff analysis
stringtie 3_sorted.bam -G dmel.gtf -o 3.gtf -v -m 100 -C 3_cov.gtf -B
#if we want just to get gtfs, then it will be enough to: stringtie 3_sorted.bam -G dmel.gtf -o 3.gtf -v -m 100

#creating a list of files for cuffmerge
touch merge.txt
nano merge.txt 
#then -> add pathes to gtf-s to merge, one line for each

#merging all created GTF files (for the same of simplicity assume they are in  /home/uploader/flies/assembly4/transcripts/<name> #folders, while fasta-s are in /home/uploader/flies/assembly
cuffmerge -o /home/uploader/flies/assembly4/transcripts -g /home/uploader/flies/assembly4/transcripts/dmel.gtf -s /home/uploader/flies/assembly4/dmel.fasta -p 4 /home/uploader/flies/assembly4/transcripts/merge.txt

###Ballgrown analysis####