Page Comparison

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# Request a 180 minute interactive node on the normal queue using our reservation
idev -m 120 -N 1 -A OTH21164 -r CoreNGS
idev -m 120 -N 1 -A TRA23004 -r CoreNGS

# Request a 120 minute idev node on the development queue 
idev -m 120 -N 1 -A OTH21164 -p development
idev -m 120 -N 1 -A TRA23004 -p development

# Using our reservation
sbatch --reseservation=CoreNGS <batch_file>.slurm

Overview

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Suppose we want to know how many alignments included insertions or deletions (indels) versus the reference. Looking at the CIGAR field definition table above, we see that insertions are denoted with the character I and deletions with the character D. We'll use this information, along with samtools view, cut and grep, to count the number of indels.

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The result should be 1.22283 %.In addition to the rate, converted to a percentage, we also output the literal percent sign ( % ).

Notes:

• print 100*6697/547664 calculates the percentage rate
• The double quotes ( " )

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• surround a literal string in awk

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• • here the percent sign ( % )
• The comma ( , ) between the number and the string says to separate the two output fields (the rate and the literal text) with whatever the default Output Field Separator (OFS) is.
  • By default,

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• • awk's Output Field Separator is a single space
    • while it's default Input Field Separator (FS) is whitespace (any number of space and Tab characters)

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So what if we want to get fancy and do all this in a one-liner command? We can use echo and backtick evaluation to put both numbers in a string, then pipe that string to awk. This time we use awk body code, and refer to the two whitespace-separated fields being passed in: total count ($1) and indel count ($2).

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awk 'BEGIN{ printf("%.2f %%\n", 100*6697/547664) }'

Filtering by location range

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cd $SCRATCH/core_ngs/samtools

# count the number of reads mapped to chromosome 2 (chrII)
samtools view -c -F 0x4 yeast_pe.sort.bam chrII

# count the number of reads mapped to chromosomes 1 or M (chrI, chrM)
samtools view -c -F 0x4 yeast_pe.sort.bam chrI chrM

# count the number of reads mapped to chromosomes 1 that overlap coordinates 1000-2000
samtools view -c -F 0x4 yeast_pe.sort.bam chrI:1000-2000

# since there are only 20 reads in the chrI:1000-2000 region, examine them individually
samtools view -F 0x4 yeast_pe.sort.bam chrI:1000-2000

# look at a subset of field for chrI:1000-2000 reads
# fields:  2=flags, 3=contig, 4=start, 5=mapping quality, 6=CIGAR, 9=insert size
samtools view -F 0x4 yeast_pe.sort.bam chrI:1000-2000 | cut -f 2-6,9

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# look at a subset of field for chrI:1000-2000 reads
#  fields: 2=flags, 3=contig, 4=start, 5=mapping quality, 6=CIGAR, 9=insert size
samtools view -F 0x4 yeast_pe.sort.bam chrI:1000-2000 | cut -f 2-6,9 | \
  awk 'BEGIN{FS=OFS="\t"}
       {$1 = sprintf("0x%x", $1); print}'

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• If a command line is continued on more than one line, the line continuation character ( \ ) is used.
• There is a sprintf (string print formatted) function in awk, just as there is in many modern programming languages.
• We use sprintf to re-format the 1st input field (here the cut flags) in hex
  
  • using the format directive (%x)
  • also adding a literal "0x" prefix to denote the numeric base is hexadecimal
• The results of sprintf are stored back into the 1st field ($1), replacing the original value.
• awk's print statement is then used with no other arguments to print all its input fields, including the changed $1.

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samtools view -c -F 0x4 -f 0x2 yeast_pe.sort.bam chrI:1000-2000

About mapping quality

Mapping qualities are quality is a measure of how likely a given sequence alignment to its reported location is correct. If a read's mapping quality is low (especially if it is zero, or mapQ 0 for short) the read maps to multiple locations on the genome (they are multi-hit or multi-mapping reads), and we can and we can't be sure whether the reported location is the correct one. These are called multi-mapping or multi-hit reads.

Aligners also differ in whether they report alternate alignments for multi-hit reads, and if they do, how many. Some things to keep in mind:

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• bwa aln (global alignment) and bowtie2 with default parameters (both --local default end-to-end mode) report at most one location for a read that maps
  
  • this will be the location with the best mapping quality and alignment
  • if a given read maps equally well to multiple locations, these aligners pick one location at random
    • bwa aln will always report a 0 mapping quality for these multi-hit reads
    • bowtie2 will report a low mapping quality (< 10), based on the complexity (information content) of the sequence
• bwa mem (local alignment) can always report more than one location for a mapped read
  • its definition of a secondary alignment is different (and a bit non-standard)
    
    • if one part of a read maps to one location and another part maps somewhere else (e.g. because of RNA splicing), the longer alignment is marked as primary and the shorter as secondary.
  • there is no way to disable reporting of secondary alignments with bwa mem.
    • but they can be filtered from the sorted BAM with -F 0x100 (secondary alignment flag = 0).
• bowtie2 can be configured to report more than one location for a mapped read
  
  • the -k N option says to report up to N alignments for a read
• most transcriptome-aware RNA-seq aligners by default report more than one location for a mapped read
  • e.g. hisat2, star, tophat2.
  • when reads are quantified (counted with respect to genes), multiply-mapped reads can be counted fractionally
    • e.g. if a read maps to 5 genes, it can be counted as 1/5 for each of the genes

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samtools view -c yeast_pe.sort.bam
samtools view -c yeast_pe.sort.filt.bam

# or to be really fancy...
echo "`samtools view -c yeast_pe.sort.bam` \
      `samtools view -c yeast_pe.sort.filt.bam`" | \
       awk '{printf "%d original reads\n", $1;
             printf "%d Q20 reads (%d read pairs)\n", $2, $2/2;
             printf "%0.2f%% high-quality reads\n", 100*$2/$1}'

Exercise: How many primary aligned reads (0x100 = 0) are in Copy the bwa_local.sort.dup.bam file?

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local alignment files here:

samtools view -F 0x104 -c bwa_local.sort.dup.bam

There are 874791 primary alignments.

samtools view -F 0x4 -f 0x100 -c bwa_local.sort.dup.bam

samtools view only pays attention to one -F or -f option, so to specify more than one flag value you need to combine them into one number. For example, combining 0x100 and 0x4 yields 0x104.

cds; cd core_ngs/samtools
cp $CORENGS/catchup/bwa_script/bwa_local.sort.dup.bam* .
cp $CORENGS/catchup/bwa_script/bwa_local.samstats.txt  .

Exercise: How many primary aligned reads (0x100 = 0) are in the bwa_local.sort.dup.bam file?

samtools view -F 0x104 -c bwa_local.sort.dup.bam

samtools view -F 0x4 -f 0x100 -c bwa_local.sort.dup.bam

Look at the statistics for the bwa mem (local) alignment in the bwa_local.samstats.txt file:

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             Aligner:       bwa
     Total sequences:   1317569
        Total mapped:   1008000 (76.5 %)
      Total unmapped:    309569 (23.5 %)
             Primary:    874791 (86.8 %)
           Secondary:    133209 (13.2 %)
          Duplicates:    428418 (42.5 %)
          Fwd strand:    502782 (49.9 %)
          Rev strand:    505218 (50.1 %)
          Unique hit:    947418 (94.0 %)
           Multi hit:     60582 (6.0 %)
           Soft clip:    543390 (53.9 %)
           All match:    328038 (32.5 %)
              Indels:      6679 (0.7 %)
             Spliced:
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       Total PE seqs:   1317569
      PE seqs mapped:   1008000 (76.5 %)
        Num PE pairs:    658784
   F5 1st end mapped:    413159 (62.7 %)
   F3 2nd end mapped:    594841 (90.3 %)
     PE pairs mapped:    384462 (58.4 %)
     PE proper pairs:    282664 (42.9 %)
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