Sam:LabNotes/Microbiome-new/2010-12-8: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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'''E.coli taxonomy''' | '''E.coli taxonomy''' | ||
--------------------- | --------------------- | ||
Domain: | Domain: Eubacteria | ||
Phylum: Proteobacteria; | Phylum: Proteobacteria; | ||
Class: Gammaproteobacteria; | Class: Gammaproteobacteria; |
Revision as of 19:20, 31 December 2010
General statiscs (whole reads) and contamination analysis (failed to aligned reads) - HC-MDA-Ecoli-SAGs-Ind1to8
Background
- Samples were prepared from -(short and long amplified E.coli SAGs -3rd test 11-07-'10)
- 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-2.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 Media:ID3-failed-to-align-L300-accession.pdf Media:ID4-failed-to-align-L300-accession.pdf Media:ID5-failed-to-align-L300-accession.pdf Media:ID6-failed-to-align-L300-accession.pdf Media:ID7-failed-to-align-L300-accession.pdf Media:ID8-failed-to-align-L300-accession.pdf
E.coli taxonomy --------------------- Domain: Eubacteria Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales; Family: Enterobacteriaceae; Genus: Escherichia. Species: E. coli
Yeast taxonomy --------------------- Super kindom: Eukaryota; Domain: Fungi; Phylum: Ascomycota; Class: Saccharomycetes; Order: Saccharomycetales; Family: Saccharomycetaceae; Genus: Saccharomyces; Species: Saccharomyces cerevisiae
Discussions
- 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.
- A certain level fail-to-align contigs also match E.coli genome (e.g. ID-3 and ID-8). It is unknown why some E.coli reads are failed to map to E.coli genome in bowtie.
- Sequencing error => mis-match >3bp => fail to meet mapping criteria in bowtie?
- Chimeric junction in these "single-end" reads?
- A certain level fail-to-align contigs also match E.coli genome (e.g. ID-3 and ID-8). It is unknown why some E.coli reads are failed to map to E.coli genome in bowtie.
- 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.
- I am thinking that maybe it's a better idea to use MEGAN metagenomics tool for analyzing these fail-to-align reads directly, which it can draw a taxonomic distribution plot easily.