Análisis genómicos y transcriptómicos con plataforma NGS

Enlaces R & Bioconductor

• Central R Archive Network (CRAN)
• RStudio
• Bioconductor

Una vez instalado R y el paquete BiocManager el siguiente codigo instalará Bioconductor:

BiocManager::install()

Para saber la version de Bioconductor que estamos usando y si los paquetes estan actualizados :

BiocManager::version()
BiocManager::valid()

Instalación paquetes Bioconductor

BiocManager::install("ShortRead")  # <- manipulate FASTQ files
BiocManager::install("Rbowtie2")  # <- align FASTQ files
BiocManager::install("Rsamtools") # <- manipulate SAM files
BiocManager::install("Rsubread")  # <- quantify alignments
BiocManager::install("DESeq2")    # <- differential gene expression

Material

• Análisis expresion Diferencial RNA-Seq : tabla muestras
• European Nucleotide Archive
  Run: SRR9336468
  Run: SRR9336469
  Run: SRR9336470
  Run: SRR9336471
  Run: SRR9336472
  Run: SRR9336473
  Run: SRR9336474
  Run: SRR9336475
  Run: SRR9336476

Descarga ficheros secuencia genómica

download.file("https://www.dropbox.com/s/4ft480eky7kghzw/Saccharomyces_cerevisiae_genome.fa.gz?dl=1", 
              destfile = "Saccharomyces_cerevisiae_genome.fa.gz")

Descarga ficheros anotación genómica

download.file("https://www.dropbox.com/s/qtaret1hrbvw2xb/Saccharomyces_cerevisiae_genome.gff3.gz?dl=1", 
              destfile = "Saccharomyces_cerevisiae_genome.gff3.gz")              

Descarga ficheros lecturas

download.file("https://www.dropbox.com/s/v06um7vt9ojdf42/NGS_MB_SRR9336468_1.fastq.gz?dl=1", 
              destfile = "1M_SRR9336468_1.fastq.gz")

download.file("https://www.dropbox.com/s/kgxfth8ra675ccu/NGS_MB_SRR9336468_2.fastq.gz?dl=1", 
              destfile = "1M_SRR9336468_2.fastq.gz")

 

Mapping Reads

• STEP 1: Create genome index for bowtie2 with bowtie2_build
• STEP 2: Align FASTQ files against indexed genome with bowtie2
• STEP 3: Convert SAM files into BAM (and indexes .bai) with asBam

code

 

Differential Expression Analysis

Paper : "Physiological responses of Saccharomyces cerevisiae to industrially relevant conditions: Slow growth, low pH, and high CO2 levels". Hakkaart X, Liu Y, Hulst M, El Masoudi A, Peuscher E, Pronk J, van Gulik W, Daran-Lapujade P. Biotechnol Bioeng. 2020 Mar;117(3):721-735. doi: 10.1002/bit.27210. Epub 2020 Jan 22.

Sample table

• STEP 1: Create genome index for bowtie2 with bowtie2_build
• STEP 2: Align FASTQ files against indexed genome with bowtie2
• STEP 3: Convert SAM files into BAM (and indexes .bai) with asBam
• STEP 4: Create metadata file for the Saccharomyces count matrix
• STEP 5 : Quantify genes with featureCounts.
• STEP 6 : Prepare data for the DESeq2 workflow.
• STEP 7 : Create a DESeq2 object with DESeqDataSetFromMatrix