Sam:LabNotes/Microbiome-new/2011-2-15: Difference between revisions

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==Background==
==Background==
*Samples were prepared from -(short and long amplified E.coli SAGs -3rd test 11-07-'10)
*Ecoli SAGs were prepared from flow-sorted single E.coli cells.
*Eight E.coli SAGs samples were made to Illumina sequencing libraries using (N2)Primer-based barcodes ID1 to ID8.
*Eight E.coli SAGs samples were made to Nextera-libraries.
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-12-29
**http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2011-01-06_exp2
 
*These libraries were equally pulled (20ng per library) and sequenced in pair-end(PE) mode.
*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.
*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.
 
Wiki(12-29-'10)
Sample no.  Sample name          Total DNA(ng)
-----------------------------------------------
NX01        WGA102710-(1-4)- 3hr    288.50ng
NX02        WGA102710-(1-4)-10hr    500.00ng
NX03        WGA110310-(5)-  3hr    114.50ng
NX04        WGA110310-(5)-  10hr    500.00ng
 
Wiki(01-06-'11)
Sample no.  Sample name          Total DNA(ng)
-----------------------------------------------
NX05        WGA122010-(1-2)-3hr      181.50ng
NX06        WGA122010-(1-2)-10hr      200.00ng
NX07        WGA122110-(2-3)-3hr        50.00ng
NX08        WGA122110-(2-3)-10hr      200.00ng


==Analysis approaches==
==Analysis approaches==

Revision as of 23:50, 26 February 2011

Bowtie mapping(whole reads) and contamination analysis(failed to aligned reads)- HC-MDA-Ecoli-SAGs(NX1-NX8)-Jan17

Background

  • 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.
Wiki(12-29-'10)

Sample no.   Sample name          Total DNA(ng)
-----------------------------------------------
NX01         WGA102710-(1-4)- 3hr     288.50ng
NX02         WGA102710-(1-4)-10hr     500.00ng
NX03         WGA110310-(5)-   3hr     114.50ng
NX04         WGA110310-(5)-  10hr     500.00ng
Wiki(01-06-'11)

Sample no.   Sample name          Total DNA(ng)
-----------------------------------------------
NX05         WGA122010-(1-2)-3hr       181.50ng
NX06         WGA122010-(1-2)-10hr      200.00ng
NX07         WGA122110-(2-3)-3hr        50.00ng
NX08         WGA122110-(2-3)-10hr      200.00ng

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
 ABySS-fac report

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 -v 1 –b 1(using -m 8 output format to extract accession number)
    • BLAST setting: -K 1 -m 8 -a 2 -v 1 –b 1(using -m 9 output format to get BLAST statistics)
      • NOTE: Use -K 1 -v 1 -b 1 for reporting only one hit
    • Taxonomic analysis by NCBI Entrez (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 taxonomic ranks
-------------------------
Domain:   Eubacteria;
Phylum:     Proteobacteria;
Class:        Gammaproteobacteria;
Order:          Enterobacteriales;
Family:            Enterobacteriaceae;
Genus:                Escherichia; 
Species:                 E. coli
Yeast taxonomic ranks - (to detect if Yeast DNA contamination was introduced through Yeast tRNA carrier)
------------------------
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.
           ID1   ID2   ID3   ID4   ID5    ID6   ID7    ID8
-----------------------------------------------------------
Homo hits   N/A    22     0    25   155     86    35     83
All  hits   N/A    75    15   106   200    145    56    120
(%)              29.3%       23.6% 77.5%  59.3%  62.5% 69.1%

All failed-to-align contig hit

  • 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?
  • 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.