🏫 Práticas da Disciplina de Bioinformática (Bioinformatics Discipline Practices)

This is a repository for the Bioinformatics Discipline practices.

👉 Setup the environment

How to install Conda?

• Download Anaconda software from https://www.anaconda.com/products/individual
• Install it (https://docs.anaconda.com/anaconda/install)

How to set up the environment using conda?

Option 01:

Download the file environment.yml

conda env create -f environment.yml

Option 02:

conda create -n pipelines python=3.6 r=3.6 -y

How to enter in the conda environment?

conda activate pipelines

How to setup the channels (repositories) with the needed tools?

conda config --add channels bioconda
conda config --add channels conda-forge

How to install the needed tools into the DisciplinaBioinfo environment?

conda install pandas numpy jupyterlab jupyter nano readline=6.2 sra-tools entrez-direct bwa fastqc fastp spades quast star htseq seqtk samtools=1.13 bcftools=1.13 r-xml freebayes bedtools vcflib rtg-tools matplotlib -y

• R packages (run it from the R prompt):

install.packages('IRkernel')

install.packages("BiocManager")

BiocManager::install(c("limma","edgeR","Glimma","data.table","org.Mm.eg.db", "statmod"))

📔 Practice 01 - De novo assembly of a Brazillian isolate of Sars-Cov-2 Genome

Enter in the conda environmet

conda activate pipelines

Obtaining the raw material

• SARS-CoV-2 genome sequencing Rio Grande do Sul / Brazil, Dec 2020; Total RNA from SARS-CoV-2 positive samples was converted to cDNA. Viral whole-genome amplification was performed according to the Artic Network. Available in SRR13510367.

fastq-dump --accession SRR13510367 --split-files --outdir rawdata -v

Generating a quality report for the sequenced reads

fastqc rawdata/SRR13510367_1.fastq rawdata/SRR13510367_2.fastq

Filtering data with fastp

mkdir filtered_data

fastp --thread 4 -p -q 30 -i rawdata/SRR13510367_1.fastq -I rawdata/SRR13510367_2.fastq -o filtered_data/SRR13510367_1_FILTERED.fastq -O filtered_data/SRR13510367_2_FILTERED.fastq &

mv fastp.* filtered_data/

Running the asembly with Spades

spades.py -t 4 -o assembly --careful -k 21,33,55 -1 filtered_data/SRR13510367_1_FILTERED.fastq -2 filtered_data/SRR13510367_2_FILTERED.fastq &

generate a report about the assembly with Quast

• Download the Sars-Cov-2 reference genome

esearch -db nucleotide -query "NC_045512.2" | efetch -format fasta > NC_045512.2.fasta

• Performe the report

quast assembly/scaffolds.fasta -R NC_045512.2.fasta

📔 Practice 02 - Transcriptome of human T cell stimulated with anti-CD3 antibody (Sousa, 2019)

Briefing: human peripheral blood mononuclear cells were purified from healthy volunteers blood and were cultured in the presence of the monoclonal antibody OKT3 or a recombinant fragment of humanized anti-CD3 (FvFcR) or recombinant fragment chimeric anti-CD3 (FvFcM).

• For the tutorial, we only will use the chromossome 22 and a monoclonal antibody OKT3 sample to make it feasible in a personal computer.

Enter in the conda environmet

conda activate pipelines

Obtaining the raw material

• Homo sapiens reference genome
• Chromossome 22 DNA sequence
• Chromossome 22 GFF3 annotation
• Chromossome 22 GTF annotation
• OKT3 replica 1 = SRR6974025
• FvFcR replica 1 = SRR6974027

Downloading the reads

fastq-dump --accession SRR6974025 --outdir rawdata -v

fastq-dump --accession SRR6974027 --outdir rawdata -v

Filtering the reads quality using fastp

fastqc rawdata/SRR6974025.fastq

fastqc rawdata/SRR6974027.fastq

mkdir filtered_data

fastp --thread 8 -p -q 30 -i rawdata/SRR6974025.fastq -o filtered_data/SRR6974025_FILTERED.fastq

fastp --thread 8 -p -q 30 -i rawdata/SRR6974027.fastq -o filtered_data/SRR6974027_FILTERED.fastq

mv fastp.* filtered_data/

Generating an index for the genome reference

mkdir genome_reference && cd genome_reference

wget http://ftp.ensembl.org/pub/release-104/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.chromosome.22.fa.gz

wget http://ftp.ensembl.org/pub/release-104/gtf/homo_sapiens/Homo_sapiens.GRCh38.104.chr.gtf.gz

gunzip Homo_sapiens.GRCh38.dna.chromosome.22.fa.gz && gunzip Homo_sapiens.GRCh38.104.chr.gtf.gz && cd ../

STAR --runThreadN 8 --runMode genomeGenerate --genomeDir genome_reference --genomeFastaFiles genome_reference/Homo_sapiens.GRCh38.dna.chromosome.22.fa --sjdbGTFfile genome_reference/Homo_sapiens.GRCh38.104.chr.gtf --sjdbOverhang 49

Mapping the filtered reads to the genome using STAR

STAR --genomeDir genome_reference --runThreadN 8 --readFilesIn filtered_data/SRR6974025_FILTERED.fastq filtered_data/SRR6974027_FILTERED.fastq --outFileNamePrefix mapped_data --outSAMtype BAM SortedByCoordinate --outSAMunmapped Within --outSAMattributes Standard --quantMode GeneCounts TranscriptomeSAM

Counting mapped genes with htseq-count

htseq-count --nonunique all --format bam --stranded no --order pos --type exon --idattr gene_id <FILE_NAME>.out.bam genome_reference/Homo_sapiens.GRCh38.104.chr.gtf > table.counts

References

Sousa IG, Simi KCR, do Almo MM, Bezerra MAG et al. Gene expression profile of human T cells following a single stimulation of peripheral blood mononuclear cells with anti-CD3 antibodies. BMC Genomics 2019 Jul 19;20(1):593. PMID: 31324145

📔 Practice 02.1 - EGF-mediated induction of Mcl-1 at the switch to lactation is essential for alveolar cell survival (Fu, 2019)

Análise de genes diferencialmente expressos em células de glândulas mamárias de camundongos fêmeas em duas situações: grávidas e lactantes. Neste tutorial, a partir de dados já mapeados e contados, é realizada a análise de gene diferencialmente expressos (DEGs).

Link par o Tutorial

📔 Practice 03 - COVID-19 variant calling from Illumina data

This practice is an adaptation of

• Nielsen R, Paul JS, Albrechtsen A, Song YS. Genotype and SNP calling from next-generation sequencing data. Nat Rev Genetics, 2011, 12:433-451.

• Olsen ND et al. Best practices for evaluating single nucleotide variant calling methods for microbial genomics. Front. Genet., 2015, 6:235.

Enter in the conda environmet

conda activate pipelines

Obtaining the raw material

• SARS-CoV-2 genome sequencing Rio Grande do Sul / Brazil, Dec 2020; Total RNA from SARS-CoV-2 positive samples was converted to cDNA. Viral whole-genome amplification was performed according to the Artic Network. Available in SRR13510367.

fastq-dump --accession SRR13510367 --outdir rawdata -v

• Download the Sars-Cov-2 reference genome

mkdir genome && esearch -db nucleotide -query "NC_045512.2" | efetch -format fasta > genome/NC_045512.2.fasta

Alignment with Map with BWA-MEM

• create the genome index

bwa index genome/NC_045512.2.fasta

• Map reads to the reference genome

bwa mem -t 4 genome/NC_045512.2.fasta rawdata/SRR13510367.fastq > SRR13510367.sam

• Convert the SAM file to BAM format

samtools view -S -b SRR13510367.sam > SRR13510367.bam

• Sort BAM file by coordinates

samtools sort -o SRR13510367_sorted.bam SRR13510367.bam

• create an sam indexes for the genome and the mapped reads (bam)

samtools faidx genome/NC_045512.2.fasta

samtools index SRR13510367_sorted.bam

• Some stats about yout alignment

samtools flagstat SRR13510367_sorted.bam

Call variants

• Create a folder to save the variant resuls

mkdir variants

• generating a file with the variants

freebayes -p 1 -f genome/NC_045512.2.fasta SRR13510367_sorted.bam > variants/SRR13510367.vcf

or (using samtools)

bcftools mpileup -Ou -f genome/NC_045512.2.fasta SRR13510367_sorted.bam | bcftools call -mv -o variants/SRR13510367.vcf

• Take a look in the first lines

cat variants/SRR13510367.vcf | grep -v '##' | head -4

Some stats

• Compressing the file

bgzip variants/SRR13510367.vcf

• Creating an index for the variants file

tabix -p vcf variants/SRR13510367.vcf.gz

• Quick stats

rtg vcfstats variants/SRR13510367.vcf.gz

• More detailed stats

bcftools stats -F genome/NC_045512.2.fasta -s - variants/SRR13510367.vcf.gz > variants/SRR13510367.vcf.gz.stats

• Plots

mkdir variants/plots

plot-vcfstats -p variants/plots/ variants/SRR13510367.vcf.gz.stats