From 5fe8ee6522ea20907c8c85dc15eff082afcefd2a Mon Sep 17 00:00:00 2001 From: AshKernow Date: Wed, 25 Sep 2024 15:12:04 +0100 Subject: [PATCH] Add the tx2gene section back in --- ...3_Quantification_with_Salmon_practical.Rmd | 33 ++++++++++++++++-- ..._Quantification_with_Salmon_practical.html | 32 +++++++++++++---- ...3_Quantification_with_Salmon_practical.pdf | Bin 89140 -> 92224 bytes 3 files changed, 57 insertions(+), 8 deletions(-) diff --git a/Markdowns/03_Quantification_with_Salmon_practical.Rmd b/Markdowns/03_Quantification_with_Salmon_practical.Rmd index d41318d..367cfa4 100644 --- a/Markdowns/03_Quantification_with_Salmon_practical.Rmd +++ b/Markdowns/03_Quantification_with_Salmon_practical.Rmd @@ -208,7 +208,7 @@ Salmon creates a separate output directory for each sample analysed. This directory contains a number of files; the file that contains the quantification data is called `quant.sf`. -### 3.1 SAM to BAM with samtools +# 3 SAM to BAM with samtools We can transform from SAM to BAM using `samtools`. `samtools` is a toolkit that provides a number of useful tools for working with SAM/BAM files. The BAM file @@ -227,7 +227,7 @@ other options, e.g. reads can be sorted by the read name instead of the position or we can specify the number of parallel threads to be used - to find out more use `samtools sort --help`. -### Exercise 3 +## Exercise 3 > 1. Sort and transform your aligned SAM file into a BAM file called > `SRR7657883.salmon.sorted.bam`. Use the option `-@ 7` to use 7 cores, this @@ -235,6 +235,35 @@ use `samtools sort --help`. > 2. Use, for example, `samtools view my_sample.sorted.bam` to check your BAM file +# 4 Make transcript to gene table + +Salmon quantifies gene expression at the transcript level. When we come to do +our differential gene expression analysis in R, we will want to summarise this +to the gene level. To do this we need a table that links transcript IDs to gene +IDs. We have already created this for you, but, for reference, the code below +was used to generate this table from the sequence headers in the transcriptome +reference file. + +**You do not need to run this code, we have already done this for you.** + +```{bash eval=FALSE} +echo -e "TxID\tGeneID" > salmon_outputs/tx2gene.tsv +zcat references/Mus_musculus.GRCm38.cdna.all.fa.gz | + grep "^>" | + head | + cut -f 1,4 -d ' ' | + sed -e 's/^>//' -e 's/gene://' -e 's/\.[0-9]*$//' | + tr ' ' '\t' \ + >> salmon_outputs/tx2gene.tsv +``` + +1. `zcat references/Mus_musculus.GRCm38.cdna.all.fa.gz` - read the zipped fasta +1. `grep "^>"` - find the sequence headers, they all start with ‘>’ +1. `cut -f 1,4 -d ' '` - extract the 1st and 4th entries on each line - transcript ID and gene ID +1. `sed -e 's/^>//' -e 's/gene://' -e 's/\.[0-9]*$//'` - remove the “>” from the beginning of the line, the “gene:” from the beginning of the gene ID, and the trailing “.x” number which indicates that version of the gene annotation +1. `tr ' ' '\t'` - replace spaces with tabs so that the table is tab delimited + + ---------------------------------------------------------- # References \ No newline at end of file diff --git a/Markdowns/03_Quantification_with_Salmon_practical.html b/Markdowns/03_Quantification_with_Salmon_practical.html index af26bc4..34ae571 100644 --- a/Markdowns/03_Quantification_with_Salmon_practical.html +++ b/Markdowns/03_Quantification_with_Salmon_practical.html @@ -272,15 +272,16 @@

Exercise 2 - Quantify with Salmon

Salmon creates a separate output directory for each sample analysed. This directory contains a number of files; the file that contains the quantification data is called quant.sf.

-
-

3.1 SAM to BAM with samtools

+
+ +
+

3 SAM to BAM with samtools

We can transform from SAM to BAM using samtools. samtools is a toolkit that provides a number of useful tools for working with SAM/BAM files. The BAM file format is a binary (not human readable) file and is considerably smaller than the same data stored in SAM format. We will also sort the alignment entries by location (Contig/Chromosome name and the location on the contig), this further improves the compression of the SAM to BAM. We will use the samtools sort function.

The general command is:

samtools sort -O BAM -o my_sample.sorted.bam my_sample.sam

Where the -o option is used to provide the output file name. There are many other options, e.g. reads can be sorted by the read name instead of the position or we can specify the number of parallel threads to be used - to find out more use samtools sort --help.

-
-
-

Exercise 3

+
+

Exercise 3

  1. Sort and transform your aligned SAM file into a BAM file called SRR7657883.salmon.sorted.bam. Use the option -@ 7 to use 7 cores, this vastly speeds up the compression.
  2. @@ -291,9 +292,28 @@

    Exercise 3

  3. Use, for example, samtools view my_sample.sorted.bam to check your BAM file
-
+
+

4 Make transcript to gene table

+

Salmon quantifies gene expression at the transcript level. When we come to do our differential gene expression analysis in R, we will want to summarise this to the gene level. To do this we need a table that links transcript IDs to gene IDs. We have already created this for you, but, for reference, the code below was used to generate this table from the sequence headers in the transcriptome reference file.

+

You do not need to run this code, we have already done this for you.

+
echo -e "TxID\tGeneID" > salmon_outputs/tx2gene.tsv
+zcat references/Mus_musculus.GRCm38.cdna.all.fa.gz |
+  grep "^>" | 
+  head |
+  cut -f 1,4 -d ' ' | 
+  sed -e 's/^>//' -e 's/gene://' -e 's/\.[0-9]*$//' |
+  tr ' ' '\t' \ 
+  >> salmon_outputs/tx2gene.tsv
+
    +
  1. zcat references/Mus_musculus.GRCm38.cdna.all.fa.gz - read the zipped fasta
  2. +
  3. grep "^>" - find the sequence headers, they all start with ‘>’
  4. +
  5. cut -f 1,4 -d ' ' - extract the 1st and 4th entries on each line - transcript ID and gene ID
  6. +
  7. sed -e 's/^>//' -e 's/gene://' -e 's/\.[0-9]*$//' - remove the “>” from the beginning of the line, the “gene:” from the beginning of the gene ID, and the trailing “.x” number which indicates that version of the gene annotation
  8. +
  9. tr ' ' '\t' - replace spaces with tabs so that the table is tab delimited
  10. +
+

References

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