Changes between Version 23 and Version 24 of SOPs/qc_shortReads

Timestamp:

04/27/16 11:20:16 (10 years ago)

Author:

ibarrasa

Comment:

--

SOPs/qc_shortReads

@@ -243 +243 @@
 = Analyzing short read quality (after mapping) =
 
-== Remove Duplicates ==
-  * Remove duplicates, for eg. from PCR
-
- {{{
-   #samtools command
-    samtools rmdup [-sS] <input.srt.bam> <output.bam>
-    -s or -S depending on PE data or not
-}}}
-
-== Determining the paired-end insert size for DNA samples ==
-
-If paired-end insert size or distance is unknown or need to be verified, it can be extracted from a BAM/SAM file after running Bowtie.  
-
-When mapping with bowtie (or another mapper), the insert size can often be included as an input parameter (example for bowtie: -X 500), which can help with mapping.  See the [[http://barcwiki.wi.mit.edu/wiki/SOPs/mapping|mapping SOP]] for mapping details.
-
-
-Method 1: Get insert sizes from BAM file
-{{{
-   # Using a SAM file (at Unix command prompt)
-   awk -F "\t" '$9 > 0 {print $9}' s_1_bowtie.sam > s_1_insert_sizes.txt
-   # Using a BAM file (at Unix command prompt)
-   samtools view s_1_bowtie.bam | awk -F"\t" '$9 > 0 {print $9}' > s_1_insert_sizes.txt
-
-   # and then process column of numbers with R (or Excel)
-   # In R Session
-   sizeFile = "s_1_insert_sizes.txt"
-   sample.name = "My paired reads"
-   distance = read.delim(sizeFile, h=F)[,1]
-   pdf(paste(sample.name, "insert.size.histogram.pdf", sep="."), w=11, h=8.5)
-   hist(distance, breaks=200, col="wheat", main=paste("Insert sizes for", sample.name), xlab="length (nt)")
-   dev.off()
-}}}
-
-Method 2: Calculate insert sizes with CollectInsertSizeMetrics function from picard (http://picard.sourceforge.net).  This is also a good approximation for RNA samples.
-{{{
-   # 
-   # I=File    Input SAM or BAM file.  (Required)
-   # O=File    File to write the output to.  (Required)
-   # H=File    File to write insert size histogram chart to.  (Required)
-   # output: CollectInsertSizeMetrics.txt: values for -r and --mate-std-dev can be found in this text file
-   #         CollectInsertSizeMetrics_hist.pdf: insert size histogram (graphic representation)
-bsub java -jar  /usr/local/share/picard-tools/CollectInsertSizeMetrics.jar I=foo.bam O=CollectInsertSizeMetrics.txt H=CollectInsertSizeMetrics_hist.pdf
-}}}
-
-== [RNA-seq only] Get global coverage profile across transcripts ==
-
-Do reads come from across the length of a typical transcript, or is there 3' or 5' bias (where most reads come from one end of a typical transcript)?
-
-One way to look at this is with Picard's CollectRnaSeqMetrics tool
-{{{
-   # Usage:
-   java -jar /usr/local/share/picard-tools/CollectRnaSeqMetrics.jar INPUT=bamFile REF_FLAT=refFlatFile STRAND_SPECIFICITY=NONE OUTPUT=outputFile REFERENCE_SEQUENCE=/path/to/genome.fa CHART_OUTPUT=output.pdf VALIDATION_STRINGENCY=SILENT
-   # Example command
-   java -jar /usr/local/share/picard-tools/CollectRnaSeqMetrics.jar INPUT=WT.bam REF_FLAT=/nfs/genomes/mouse_mm10_dec_11_no_random/anno/refFlat.txt STRAND_SPECIFICITY=NONE OUTPUT=QC_metrics/WT.RnaSeqMetrics.txt REFERENCE_SEQUENCE=/nfs/genomes/mouse_mm10_dec_11_no_random/fasta_whole_genome/mm10.fa CHART_OUTPUT=QC_metrics/WT.RnaSeqMetrics.pdf VALIDATION_STRINGENCY=SILENT
-
-}}}
-The VALIDATION_STRINGENCY=SILENT option will keep the program from crashing if it finds something unexpected.  The default: VALIDATION_STRINGENCY=STRICT
-
-
-= Interpreting quality control issues =
-
-See [[https://sequencing.qcfail.com/|QC Fail Sequencing]] from the Babraham Institute
+See [[http://barcwiki.wi.mit.edu/wiki/SOPs/SAMBAMqc|SAM/BAM quality control]]
+
+