Changes between Version 2 and Version 3 of SOPs/rna-seq-diff-expressions_TE


Ignore:
Timestamp:
02/23/21 16:37:47 (4 years ago)
Author:
twhitfie
Comment:

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  • SOPs/rna-seq-diff-expressions_TE

    v2 v3  
    1 == Using RNA-Seq to quantify gene levels and assay for differential expression for transposable elements ==
     1== Using RNA-Seq to quantify gene levels and assay for differential expression including transposable elements ==
    22
    33=== Background ===
    … …  
    99  * **Mapping**
    1010    * Use [https://github.com/alexdobin/STAR STAR] or another spliced mapper to map short reads to the genome of choice.
    11     * See our [http://barcwiki.wi.mit.edu/wiki/SOPs/mapping mapping SOP] for more details.
     11    * See our [http://barcwiki.wi.mit.edu/wiki/SOPs/mapping mapping SOP] to search for details on running STAR.
    1212
    1313  * **Quantification of raw counts**
    1414
    15     * Is your sequencing library stranded or unstranded?  This information is needed to help these tools accurately count features.  Strandedness of some library prep methods:
    16       * TruSeq Stranded mRNA Kits ("TruSeqStrandedPolyA") reads are reverse stranded (stranded in the reverse direction relative to the transcript orientation).
    17       * SMART-Seq v4 Ultra Low Input RNA Kit ("SMARTerUltra-lowPOLYA-V4") reads are unstranded.
    18       * KAPA RNA HyperPrep Kits ("KAPAHyperPrepmRNA") reads are reverse stranded.
    19       * The Whitehead Genome Core has some more [http://genomecore.wi.mit.edu/index.php/NCBISubmission Library Prep Descriptions].
    20     * See [[SAMBAMqc]] (and/or look at mapped reads in a genome browser) to determine or verify strandedness
     15{{{
     16# Unstranded reads
     17TEtranscripts --format BAM --stranded no -t treat1.bam treat2.bam -c control1.bam control2.bam --GTF genes.gtf --TE transposons.gtf --mode multi --project treat_vs_control --minread 1 -i 100 --padj 0.05 --norm DESeq_default --sortByPos
    2118
    22 {{{
    23 # single-end reads (unstranded)
    24 featureCounts -a gene_anotations.gtf -o MySample.featureCounts.txt MySample.bam
    25 # single-end reads (forward stranded)
    26 featureCounts -s 1 -a gene_anotations.gtf -o MySample.featureCounts.txt MySample.bam
    27 # single-end reads (reverse stranded)
    28 featureCounts -s 2 -a gene_anotations.gtf -o MySample.featureCounts.txt MySample.bam
     19# Forward stranded reads
     20TEtranscripts --format BAM --stranded forward -t treat1.bam treat2.bam -c control1.bam control2.bam --GTF genes.gtf --TE transposons.gtf --mode multi --project treat_vs_control --minread 1 -i 100 --padj 0.05 --norm DESeq_default --sortByPos
    2921
    30 
    31 # paired-end reads (unstranded)
    32 featureCounts -p -a gene_anotations.gtf -o MySample.featureCounts.txt MySample.bam
    33 # paired-end reads (forward stranded)
    34 featureCounts -p -s 1 -a gene_annotations.gtf -o MySamples.featureCounts.txt *sortedByName.bam
    35 # paired-end reads (reverse stranded)
    36 featureCounts -p -s 2 -a gene_annotations.gtf -o MySamples.featureCounts.txt *sortedByName.bam
     22# Reverse stranded reads
     23TEtranscripts --format BAM --stranded reverse -t treat1.bam treat2.bam -c control1.bam control2.bam --GTF genes.gtf --TE transposons.gtf --mode multi --project treat_vs_control --minread 1 -i 100 --padj 0.05 --norm DESeq_default --sortByPos
    3724}}}
    3825
    3926
    4027 * **Other**
    41    * Review articles:
    42       * [[http://www.ncbi.nlm.nih.gov/pubmed/24020486|Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data.]] - Rapaport F, Khanin R, Liang Y, Pirun M, Krek A, Zumbo P, Mason CE, Socci ND, Betel D.  Genome Biol. 2013 Sep 10;14(9):R95.
    43       * [[http://www.ncbi.nlm.nih.gov/pubmed/21106489|A survey of statistical software for analyzing RNA-seq data]] - Gao D, Kim J, Kim H, Phang TL, Selby H, Tan AC, Tong T.  Hum Genomics. 2010 Oct;5(1):56-60.
    44       * [[http://www.ncbi.nlm.nih.gov/pubmed/21176179|From RNA-seq reads to differential expression results]] - Oshlack A, Robinson MD, Young MD.  Genome Biol. 2010;11(12):220. Epub 2010 Dec 22.
    45    * For more practical information, see the third session of [http://jura.wi.mit.edu/bio/education/R2011/ An introduction to R and Bioconductor: A BaRC Short Course] and the  [http://jura.wi.mit.edu/bio/hot_topics/ BaRC Hot Topic] (under "Short Read Sequencing", see "Practical RNA-Seq analysis")
     28   * Alternative software:
     29      * [[https://www.nature.com/articles/s41467-019-13035-2|Transposable element expression in tumors is associated with immune infiltration and increased antigenicity]] -  Yu Kong, Christopher M. Rose, Ashley A. Cass, Alexander G. Williams, Martine Darwish, Steve Lianoglou, Peter M. Haverty, Ann-Jay Tong, Craig Blanchette, Matthew L. Albert, Ira Mellman, Richard Bourgon, John Greally, Suchit Jhunjhunwala & Haiyin Chen-Harris ''Nature Communications'' '''10''', 5228 (2019)
    4630