Page Comparison

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idev -m 120 -N 1 -A OTH21164 -r CoreNGS-Fri     # or -A TRA23004
# or
idev -m 90 -N 1 -A OTH21164 -p development  # or -A TRA23004

Copy over the yeast RNA-seq files we'll need (also copy the GFF gene annotation file if you didn't make one).

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module load samtools

cd $SCRATCH/core_ngs/bedtools_multicov
samtools flagstat yeast_mrna.sort.filt.bam | tee yeast_mrna.flagstat.txt

3347559 + 0 in total (QC-passed reads + QC-failed reads)
24317 + 0 secondary
0 + 0 supplementary
922114 + 0 duplicates
3347559 + 0 mapped (100.00% : N/A)
3323242 + 0 paired in sequencing
1661699 + 0 read1
1661543 + 0 read2
3323242 + 0 properly paired (100.00% : N/A)
3323242 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

samtools view yeast_mrna.sort.filt.bam | cut -f 5 | sort | uniq -c 

    498 20
   6504 21
   1012 22
    355 23
   1054 24
   2800 25
    495 26
  14133 27
    282 28
    358 29
    954 30
   1244 31
    358 32
   6143 33
    256 34
    265 35
   1112 36
    905 37
    309 38
   4845 39
   5706 40
    427 41
   1946 42
   1552 43
   1771 44
   6140 45
   1771 46
   3049 47
   3881 48
   3264 49
   4475 50
  15692 51
  25378 52
  16659 53
  18305 54
   7108 55
   2705 56
  59867 57
   2884 58
   2392 59
3118705 60

samtools view yeast_mrna.sort.filt.bam | awk '
  BEGIN{FS="\t"; sum=0; tot=0}
  {sum = sum + $5; tot = tot + 1}
  END{printf("mapping quality average: %.1f for %d reads\n", sum/tot,tot) }'

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idev -m 120 -N 1 -A OTH21164 -r CoreNGSday5 CoreNGS  # or -A TRA23004
module load biocontainers
module load samtools
module load bedtools

mkdir -p $SCRATCH/core_ngs/bedtools_multicov
cd $SCRATCH/core_ngs/bedtools_multicov
cp $CORENGS/catchup/bedtools_merge/merged*bed .
cp $CORENGS/yeast_rnaseq/yeast_mrna.sort.filt.bam* .

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wc -l yeast_mrna_gene_counts.bed

Exercise: How many features have non-zero overlap counts?

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A signal track is a bedGraph (BED3+) file with an entry for every base in a defined set of regions that shows the count of overlapping bases for the regions (see https://genome.ucsc.edu/goldenpath/help/bedgraph.html). bedGraph files can be visualized in the Broad's IGV (Integrative Genomics Viewer) application (https://software.broadinstitute.org/software/igv/download) or in the UCSC Genome Browser (https://genome.ucsc.edu/).

• Go to the UCSC Genome Browser: https://genome.ucsc.edu/
• Select Genomes from the top menu bar
• Select Human from POPULAR SPECIES
  • under Human Assembly select Feb 2009 (GrCh37/hg19)
  • select GO
• In the hg19 browser page, the Layered H3K27Ac track is a signal track
  • the x-axis is the genome position
  • the y-axis represents the count of ChIP-seq reads that overlap each position
    • where the ChIP'd protein is H3K27AC (histone H3, acetylated on the Lysine at amino acid position 27)

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idev -m 120 -N 1 -A OTH21164 -r CoreNGS-day5     # or -A TRA23004
# or
idev -m 90 -N 1 -A OTH21164 -p development  # or -A TRA23004

module load biocontainers
module load bedtools

mkdir -p $SCRATCH/core_ngs/bedtools_genomecov
cd $SCRATCH/core_ngs/bedtools_genomecov 
cp $CORENGS/catchup/bedtools_merge/merged*bed .
cp $CORENGS/yeast_rnaseq/yeast_mrna.sort.filt.bam* .

Then calling bedtools genomecov is easy. The -bg option says to report the depth in bedGraph format.

cd $SCRATCH/core_ngs/bedtools_genomecov
bedtools genomecov -bg -ibam yeast_mrna.sort.filt.bam > yeast_mrna.genomecov.bedGraph

wc -l yeast_mrna.genomecov.bedGraph # 1519274 lines

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Because this bedGraph file is for the small-ish (12Mb) yeast genome, and for reads that cover only part of that genome, it is not too big – only ~34M. But depending on the species and read depth, bedGraph files can get very large, so there is a coresponding corresponding binary format called bigWig (see https://genome.ucsc.edu/goldenpath/help/bigWig.html). The program to covert a bedGraph file to bigWig format is part of the UCSC Tools suite of programs. Look for it with module spider, and note that you can get information about all the tools in it using module spider with a specific container version:

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Looking at the help for bedGraphToBigWig, we'll need a file of chromosome sizes. We can create one from our BAM header, using a Perl substitution script, which I prefer to sed(see Tips and tricks#perlpatternsubstitution):

module load ucsc_tools

cd $SCRATCH/core_ngs/bedtools_genomecov
bedGraphToBigWig  # look at its usage

# create the needed chromosome sizes file from our BAM header
module load samtools
samtools view -H yeast_mrna.sort.filt.bam | grep -P 'SN[:]' | \
  perl -pe 's/.*SN[:]//' | perl -pe 's/LN[:]//' > sc_chrom_sizes.txt

cat sc_chrom_sizes.txt

# displays:
chrI    230218
chrII   813184
chrIII  316620
chrIV   1531933
chrV    576874
chrVI   270161
chrVII  1090940
chrVIII 562643
chrIX   439888
chrX    745751
chrXI   666816
chrXII  1078177
chrXIII 924431
chrXIV  784333
chrXV   1091291
chrXVI  948066
chrM    85779

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cd $SCRATCH/core_ngs/bedtools_genomecov
export LC_COLLATE=C  # may or may not need this...
sort -k1,1 -k2,2n yeast_mrna.genomecov.bedGraph > yeast_mrna.genomecov.sorted.bedGraph
bedGraphToBigWig yeast_mrna.genomecov.sorted.bedGraph sc_chrom_sizes.txt yeast_mrna.genomecov.bw

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