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RNA- Seq Data Analysis

RNA- Seq Data Analysis. UND Genomics Core. RNA- Seq Analysis Pipeline. Sequence Quality Control Assessment. Adapter Trimming. Alignment. Quantification. Differential expression. Software For RNA- Seq Analysis. Fastq files. Contains sequence and quality information. Q-score.

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RNA- Seq Data Analysis

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  1. RNA-Seq Data Analysis UND Genomics Core

  2. RNA-Seq Analysis Pipeline Sequence Quality Control Assessment Adapter Trimming Alignment Quantification Differential expression

  3. Software For RNA-Seq Analysis

  4. Fastq files • Contains sequence and quality information

  5. Q-score • Q-score is a metric to assess the accuracy of sequencing • Relates to the probability of a wrong base call via logarithmic function. • -10log10(P)

  6. Sequence Quality Assessment Fastqc–o FastQC *fastq.gz Good data Bad data

  7. Sequence Quality Assessement • Q-scores based with respect to the location on flow cell • Ideally should be all blue

  8. Sequence Quality Assessement • Common for RNA-Seq • Not all FastQC warnings apply

  9. Adapter contamination Read1 cDNA adapter

  10. Adapter Trimming cDNA • Trim galore • Tries to automatically detect adapter • For Illumina adapters, the first 13 bases of the Illumina indexed adapters What trim galore matches

  11. RNA-Seq Analysis Pipeline Sequence Quality Assessment Adapter Trimming Alignment Quantification Differential expression

  12. Alignment for RNA-Seq • For eukaryotic genomes, splice- aware aligners can align reads across exon – intron boundaries. Spliced read

  13. Splice – Aware Alignment Programs • Tophat - https://ccb.jhu.edu/software/tophat/index.shtml • Hisat2 - https://ccb.jhu.edu/software/hisat2/manual.shtml • STAR - https://github.com/alexdobin/STAR

  14. Non Splice-aware Aligners • Bowtie • http://bowtie-bio.sourceforge.net/index.shtml • Bowtie2 • http://bowtie-bio.sourceforge.net/bowtie2/index.shtml • BWA • http://bio-bwa.sourceforge.net/

  15. Reference Files for Alignment • fasta • gtf (gene transfer format) Where to find reference files? • UCSC • http://hgdownload.cse.ucsc.edu/downloads.html • Ensembl • https://uswest.ensembl.org/downloads.html) • iGenomes • https://support.illumina.com/sequencing/sequencing_software/igenome.html

  16. More File Formats • Fasta • Indexes (*ht.1) • Binary version of the genome which allows for quick reading of the genome

  17. GTF file

  18. Alignment Output • Sam (Sequence alignment format) • Bam (binary sam file)

  19. RNA-Seq Analysis Pipeline Sequence Quality Assessment Adapter Trimming Alignment Quantification Differential expression

  20. Counting Reads • Count reads that unambiguously align to a gene • Programs that will count reads: • FeatureCounts • HTSeq Read Read Gene A Gene A Gene B Gene B

  21. Transcriptome assembly • Assemble a map of the genes in the transcriptome using the reads in present sample • Can be guided with a gtf, if using a well –annotated genome • Assembles transcriptome and estimate the abundance of transcript at the same time • Programs that will assemble transcriptomes: • Cufflinks • Stringtie

  22. Your Turn! • Work through a RNA-Seq analysis described in the file Day2_HandsOn.docx in the Day2 workshop material folder

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