update alignment QC

_posts/0002-06-01-Alignment_QC.md

@@ -37,7 +37,9 @@ Try filtering the BAM file to require or exclude certain flags. This can be done
 
 * [http://broadinstitute.github.io/picard/explain-flags.html](http://broadinstitute.github.io/picard/explain-flags.html)
 
-Try requiring that alignments are 'paired' and 'mapped in a proper pair' (=3). Also filter out alignments that are 'unmapped', the 'mate is unmapped', and 'not primary alignment' (=268)
+Try requiring that alignments are 'paired' and 'mapped in a proper pair' (=3). 
+
+Also filter out alignments that are 'unmapped', the 'mate is unmapped', and 'not primary alignment' (=268)
 
 ```bash
 samtools view -f 3 -F 268 UHR.bam | head | column -t | less -S
@@ -89,7 +91,7 @@ mv *fastqc.zip fastqc/
 ### Using Picard
 You can use Picard to generate RNA-seq specific quality metrics and figures
 
-In this section we need to create some additional formats of our reference files. 
+In this section we need to create some additional formats of our reference transcriptome files. 
 
 Picard uses a "sequence dictionary" file for many commands (simply a list of reference sequences and their sizes)
 
@@ -105,14 +107,15 @@ cd $RNA_HOME/refs
 # Create a .dict file for our reference
 java -jar $PICARD CreateSequenceDictionary -R chr22_with_ERCC92.fa -O chr22_with_ERCC92.dict
 
-# Create a bed file of the location of ribosomal sequences in our reference (first extract from the gtf then convert to bed)
+# Create a bed file of the location of ribosomal sequences in our reference (first extract them from the GTF then convert to BED format)
 # Note that here we pull all the "rrna" transcripts from the GTF. This is a good strategy for the whole transcriptome ...
 # ... but on chr22 there is very little "rrna" content, leading to 0 coverage for all samples, so we are also adding a single protein coding ribosomal gene "RRP7A" (normally we would not do this)
-# Note that for the convert2bed command we will treat out GTF file as a GFF file to retain exon level features when converting it to BED
+
+# Note the convert2bed command will convert our GTF to BED format
 # "<" is used to feed the GTF file into the tool.  ">2/dev/null" is used to throw away a harmless warning. "1>" is use to save our result to a file
 
 grep --color=none -i -P "rrna|rrp7a" chr22_with_ERCC92.gtf > ref_ribosome.gtf
-convert2bed --input=gff --output=bed < ref_ribosome.gtf 2>/dev/null 1>ref_ribosome.bed
+convert2bed --input=gtf --output=bed < ref_ribosome.gtf 2>/dev/null 1>ref_ribosome.bed
 
 # Create interval list file for the location of just the ribosomal sequences in our reference
 java -jar $PICARD BedToIntervalList -I ref_ribosome.bed -O ref_ribosome.interval_list -SD chr22_with_ERCC92.dict