Changes between Version 21 and Version 22 of SOPs/qc_shortReads

Timestamp:

04/26/16 09:29:51 (10 years ago)

Author:

gbell

Comment:

--

SOPs/qc_shortReads

@@ -3 +3 @@
 \\
 
-= Analyzing short read quality =
+= Analyzing short read quality (before mapping) =
 
 \\
@@ -39 +39 @@
 {{{
    bsub “/nfs/BaRC_Public/BaRC_code/Perl/cmpfastq/cmpfastq.pl s_8_1_sequence.txt s_8_2_sequence.txt”  # fastq inputs
-}}}
-
-== 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
 }}}
 
@@ -234 +199 @@
 }}}
 
-== 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
-}}}
-
 == Select reads that are paired [for paired-end sequencing]  ==
 
@@ -285 +241 @@
 }}}
 
+= 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
+}}}
+
 = Interpreting quality control issues =