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ngs-project / RNA_seq

RNA_seq differential expression
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assembled the E. coli X genome that cause deadly blood disease. #5

Closed Saraelattar closed 4 years ago

Saraelattar commented 4 years ago

[Data Retrieval](url) NCBI’s fastq-dump from sra-toolkit was used to download the short reads for NCBI short read archive (SRA).

Using SRA-toolkit

 prefetch SRR292678
  fastq-dump --outdir fastq --gzip --skip-technical  --readids --read-filter pass --dumpbase --split-3 --clip SRR292678.sra

Prepare the referance data

mkdir -p ~/workdir/sample_data
   cd ~/workdir/sample_data

wget GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_cds_from_genomic.fna.gz
 cp GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.fna GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.fa

 ` wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/221/885/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.gtf

Indexing ****``` mkdir -p ~/workdir/hisat_align/hisatIndex cd ~/workdir/hisat_align/hisatIndex

ln -s ~/workdir/sample_data/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.fa

hisat2_extract_splice_sites.py ~/workdir/sample_data/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.gtf

hisat2_extract_splice_sites.py ~/workdir/sample_data/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.gtf > splicesites.tsv

less SGCF-000221885.1_E.coli_0104_H4_illumina_1.0_genomic.fna

ln -s ~/workdir/sample_data/ ln -s ~/workdir/sample_data/SGCF-000221885.1_E.coli_0104_H4_illumina_1.0_genomic.fna .

 [ install Hisat](url)
#try to install hisat2 in linux

sudo apt_get install hisat2 sudo apt install hisat2

mkdir -p ~/workdir/hisat_align/hisatIndex && cd ~/workdir/hisat_align/hisatIndex hisat2_extract_splice_sites.py ~/workdir/sample_data/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.gtf > splicesites.tsv hisat2_extract_splice_sites.py hisat2_extract_splice hisat2 --help

(Troubleshooting)


[
 install BWA on a Linux](url)
  bunzip2 bwa-0.5.9.tar.bz2
  bunzip2 bwa-0.7.17.tar.bz2 
   tar xvf bwa-0.5.9.tar
  tar xvf bwa-0.7.17.tar
  cd bwa-0.5.9
  cd bwa-0.7.17
    make
 export PATH=$PATH:/Downloads/to/bwa-0.7.17 
   source ~/.bashrc (troubleshooting)

[*install bwa](url)

sudo apt-get install bwa

[**index my genome**](url)

mkdir -p ~/workdir/bwa_align/bwaIndex cd ~/workdir/bwa_align/bwaIndex

ln -s ~/workdir/sample_data/SGCF-000221885.1_E.coli_0104_H4_illumina_1.0_genomic.fna . bwa index -a bwtsw GCF-000221885.1_E.coli_0104_H4_illumina_1.0_genomic.fna

[[**[sequence](url) alignment](url)**](url)
 cd ~/workdir/bwa_align
  R1="$/home/crizma/Desktop/ngs/project e.coli/NEW R1 & R2/fastq/SRR292678_pass_1.fastq.gz"
  R2="$/home/crizma/Desktop/ngs/project e.coli/NEW R1 & R2/fastq/SRR292678_pass_2.fastq.gz"
    /usr/bin/time -v bwa mem bwaIndex/GCF_000221885.1_E.coli_0104_H4_illumina_1.0_genomic.fna $R1 $R2 > /SRR292678.sam
   bwa mem bwaIndex/GCF_000221885.1_E.coli_0104_H4_illumina_1.0_genomic.fna $R1 $R2 > /SRR292678.sam
  bwa mem bwaIndex/GCF_000221885.1_E.coli_0104_H4_illumina_1.0_genomic.fna $R1 $R2 > SRR292678.sam
    ls
-the sam file was empty (troubleshooting)

cd ~/workdir/bwa_align R1="$HOME/workdir/sample_data/SRR292678_pass_1.fastq.gz" cat $R1 ln -s ~/workdir/sample_data/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.fa . bwa index -a bwtsw GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.fa cd ~/workdir/bwa_align R1="$HOME/workdir/sample_data/SRR292678_pass_1.fastq.gz" R2="$HOME/workdir/sample_data/SRR292678_pass_2.fastq.gz" 

  bwa mem bwaIndex/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.fa $R1 $R2 > SRR292678.sam
  /usr/bin/time -v bwa mem bwaIndex/GCF_000221885.1_E.coli_0104_H4_Illumina_1.0_genomic.fa $R1 $R2 > SRR678.SAM

  head -n10 SRR678.SAM 
  head -n100 SRR678.SAM 
   head -n200 SRR678.SAM 

conda install -c conda-forge -y biopython
    conda install -y samtools
[   # install Samtools](url)
  ` sudo apt install samtools`
 # convert SAM file to BAM
 `samtools view -S -b SRR678.SAM -o SRR678.bam

# Sorting the BAM file

samtools sort SRR678.bam -o sorted_SRR678.bam
  head -n10 sorted_SRR678.bam

# Indexing the BAM file

`samtools index sorted_SRR678.bam`

in this point i stoped this project as i join to another group

Saraelattar commented 4 years ago

using the bioinformatic tools investigating the outbreak by assembling the genome of the deadly E. coli X strain. Specifically, we will provide you with Illumina reads from the TY2482 sample, which were generated at Beijing Genome Institute and deposited into the Short Read Archive (SRA) for public access.