Sam:LabNotes/Microbiome-new/2010-12-10: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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**Take the log10 value of total reads for Y-axis. | **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"'''. | **'''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 | ***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== | ==Results== |
Revision as of 19:38, 15 December 2010
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.