Sam:LabNotes/Microbiome-new/2010-12-10

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Revision as of 19:45, 15 December 2010 by >Sam Chiang (→‎Results)
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Data analysis- HC-MDA-Ecoli-SAGs-Ind1to8(SE - down-sampled reads) - Unique mappable reads distribution

Background

  • Genome coverage is just one indication to assess the amplification bias.
  • It is more informative to evaluate the mappable reads distribution from each library. A more even distribution (across E.coli genome) of mappable reads should be expected to see on samples with less amplification bias.

Analysis

  • Running bowtie alignment on previous down-sampled datasets and transform the output to sam-format
    • Using samtools to convert the output from sam-format to bam-format and then to pileup-format.
    • Script: fastq2pileup-hc2
    • Data resource: down-sampled raw reads (meet 1,000,000 unique mappable reads): e.g. s_4_ID1_cut_2262955.txt
    • Template:E.coli-k12-mg1655 template
    • Bowtie setting: -k 1 -l 28 -n 2 -m 1 --best --best --phred64-quals
    • Output the data: e.g. s_4_ID1_cut_2262955.bowtie.pileup
  • In UNIX, extract data from column 1, 2 and column9 with "^" symbol ("^" read starting sign in pileup-format) from each line
    • Output the data: e.g. s_4_ID1_cut_2262955.bowtie.pileup.readstart
  • Using perl to calculate "^" symbol in defined bin size (10,000 bp)
    • Script: CountReadStart-hc2
    • Output the data: e.g. s_4_ID1_cut_2262955.bowtie.pileup.readstart.count
  • Use excel to plot the Read-start counts vs. bin numbers (genome position)

Plotting

    • Since not the bin number data is not continuous (the bin number containing no reads will not be reported in previous step).
    • I need to create a bin-ladder (from bin 0 to 463) and pool my data set with bin-ladder by matching bin number.
      • Fail to do this step will cause that Excel draw the plot with discontinuous bin interval (since Excel regard bin number as a new data set but not X-axis).
    • I pooled the data set using Excel Add-Ins function "Join (merge)Talbes"(from DigDB).
    • Take the log10 value of total reads for Y-axis.
    • IMPORTANT: Since log10 value can't be take from no read (0 read), I manually assign the value of log10 of "no read data" as "-1".
      • The reason to do so is that I noticed that there are lots of bins containing "0 read" in long-amplified SAGs' data sets.

Results

File:Sam121510-read-distribution - ID1 - ID3.bmp  File:Sam121510-read-distribution - ID1 - ID3-pooled.bmp 

File:Sam121510-read-distribution - ID2 - ID4.bmp  File:Sam121510-read-distribution - ID2 - ID4-pooled.bmp 

File:Sam121510-read-distribution - ID5 - ID7.bmp  File:Sam121510-read-distribution - ID5 - ID7-pooled.bmp 

File:Sam121510-read-distribution - ID6 - ID8.bmp  File:Sam121510-read-distribution - ID6 - ID8-pooled.bmp
Excel files:
Media:Bias-plot-all-pooled ID1-ID3.xls
Media:Bias-plot-all-pooled ID2-ID4.xls
Media:Bias-plot-all-pooled ID5-ID7.xls
Media:Bias-plot-all-pooled ID6-ID8.xls