Sam:LabNotes/Microbiome-new/2010-12-8: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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[[File:sam120810-failed to aligned ID5-8.jpg|px300]] | [[File:sam120810-failed to aligned ID5-8.jpg|px300]] | ||
*Details of blast hits (PDF) | *'''Details of blast hits (PDF)''' | ||
[[Media:ID2-failed-to-align-L300-accession.pdf]] | |||
'''E.coli taxonomy''' | '''E.coli taxonomy''' | ||
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Genus: Escherichia. | Genus: Escherichia. | ||
Species: E. coli | Species: E. coli | ||
===Conclusions=== | ===Conclusions=== |
Revision as of 22:26, 13 December 2010
Data analysis of E.coli SAGs sequencing results(short and long amplified E.coli SAGs -3rd test 11-07-'10)
Background
- Eight E.coli SAGs samples were made to Illumina sequencing libraries using (N2)Primer-based barcodes ID1 to ID8.
- These libraries were equally pulled (20ng per library) and sequenced in pair-end(PE) mode.
- The sequenced data processed by Dr. Zhang for base-calling and saved into FASTQ files.
- Since I don't have genome-tech server permission, I asked Dinh's help to transfer data into Genemapster server.
Analysis approaches
Single-end data analysis
- Whole reads alignment using Bowtie => genome coverage calculate
- Script: Read2coverage-kz-3
- Data resource: HC-MDA-Ecoli-SAGs-Ind1to8
- Template:E.coli-k12-mg1655 template
- Bowtie setting: -k 1 -l 28 -n 2 -m 1 --best --best --phred64-quals
- Downstream: Data was used for genome coverage calculation.
File:Sam120610-wholereadbotie.jpg
- Using the same alignment processing, I extract the failed-to-aligned and non-specific reads into different folder.
- Script: Read2coverage-kz4
- Data resource: HC-MDA-Ecoli-SAGs-Ind1to8
- Template:E.coli-k12-mg1655 template
- Bowtie setting: -k 1 -l 28 -n 2 -m 1 --best --best --phred64-quals --un [failed-to-aligned file output] --max [nonspecific file output]
- ABySS de novo assembly of failed-to-align reads with (using single-end assembly with k-mer 27)
- Using abyss-fac function to calculate statistics
File:Sam120610-abyss assembly of fail-to-aligne reads.jpg
- Leng filtering and keep only >300bp contigs.
- Script: seqLenFilger.pl
- Perform BLAST on filtered contigs
- BLAST setting: -K 1 -m 8 -a 2 (using -m 8 output format to extract accession number)
- BLAST setting: -K 1 -m 8 -a 2 (using -m 9 output format to get BLAST statistics)
- Taxonomic analysis by NCBI Entrex (against nucletide database)
Results
px300 px300
- Details of blast hits (PDF)
Media:ID2-failed-to-align-L300-accession.pdf
E.coli taxonomy --------------------- Domain: Eubacteria Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales; Family: Enterobacteriaceae; Genus: Escherichia. Species: E. coli
Conclusions
- Most of fail-to-align reads can only be assembled to short contigs (under k-mer 27)
- The contigs assembled from fail-to-aligned reads(against E.coli template) contain a significant amount of human DNA contigs.
- ID-1 showed no BLAST hit using length>300 contigs. I tried using length>200 contigs for BLAST but it still came out no hit.
- Perform whole reads Bowtie-mapping (against E.coli-k12-mg1655 template) => analyze the failed-to-aligned reads
- Using the whole reads mapping script ()
Extracted the failed-to-aligned reads. **Perform ABySS De Novo assembly **Filter out < 300bp contigs **Local Blast, Annotating the Blast results.(folder 3) 2. Using the whole reads: Using the bowtie to do PE alignemt reads and figure out chimeric detection. Downsampling the whole reads to where total specific (output=1) E.coli alignment reaches 1,000,000 reads 3. Using the down-sampled reads: To re-calculate the new genome coverage rate using the down-sampled reads number (folder 2)(This is to test the bias extent) 4. Using the down-sampled reads: To draw bias-extent plot(This is to test the bias extent) (folder 4)
Pair-end data analysis 1. Perform ABySS De Novo assembly, compare N50, Adjust K-mer 2. Align AbySS contigs back to E.coli template using bowtie to assess mapping rate & coverage 3. Filter the read to >300bp and BLAST to nt.PE alignment using Bowtie to assess chimerics