Sam:LabNotes/Microbiome-new/2010-12-8

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Revision as of 22:26, 13 December 2010 by >Sam Chiang (→‎Results)
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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