Sam:LabNotes/Microbiome-new/2011-2-15

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Revision as of 02:08, 28 February 2011 by >Sam Chiang (→‎General genome coverage analysis)
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Bowtie mapping(whole reads) and contamination analysis(failed to aligned reads)- HC-MDA-Ecoli-SAGs(NX1-NX8)-Jan09

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

Data pooling

  • Using "gunzip" command to unzip SE files.
  • Combine two SE data(read1 + read2) into one PE data
e.g. samchiang@genemapster:~/Raw-Read-backup/110120_HL087$ cat s_4_1_ID8.txt s_4_2_ID8.txt > s_4_pe_ID8.txt &


Genome coverage analysis (whole reads and 1000000 down-sampled reads

    • Script: Read2coverage-kz-3
    • Data resource: pooled pe reads data from HC-MDA-Ecoli-SAGs(NX1-NX8)-Jan09
    • Command
perl Read2coverage-kz-3 /media/disk-2/samchiang/Raw-Read-backup/110120_HL087/pooled_pe/s_4_pe_ID8_3074085.txt /media/disk-2/samchiang/Tools/bowtie-0.12.5/indexes/e_coli_k12_mg1655/e_coli_k12_mg1655 &
    • Bowtie setting
samchiang@genemapster:~/Scripts$ /media/disk-2/samchiang/Tools/bowtie-0.12.5/bowtie -k 1 -l 28 -n 2 -m 1 --best --best --phred64-quals 
Sequence Read Name s_4_pe_ID1_NX s_4_pe_ID2_NX s_4_pe_ID3_NX s_4_pe_ID4_NX s_4_pe_ID5_NX s_4_pe_ID6_NX s_4_pe_ID7_NX s_4_pe_ID8_NX
Library Nick Name NX01 NX02 NX03 NX04 NX05 NX06 NX07 NX08
Library Sample Name WGA102710_(1_4)_3hr WGA102710_(1_4)_10hr WGA102710_(5)_3hr WGA102710_(5)_10hr WGA122010_(1-2)_3hr WGA122010_(1-2)_10hr WGA122010_(2-3)_3hr WGA122010_(2-3)_10hr
DNA starting amount (ng) 288.5 500.0 114.5 500.0 181.5 200.0 50.0 200.0
Proccessed reads 13395058 8843072 6451756 15366518 5071132 7353034 6647566 4888674
Mapped read, no. (unique) 2707193 (20.21%) 5734751 (64.85%) 1988199 (30.82%) 8571871 (55.78%) 742103 (14.63%) 2963238 (40.30%) 1048466 (15.77%) 1590418 (32.53%)
Failed to align, no. 10515640 (78.50%) 2512119 (28.41%) 4285220 (66.42%) 4736204 (30.82%) 4311962 (85.03%) 4338695 (59.01%) 5549873 (83.49%) 3176843 (64.98%)
Mapped read, no (non-unique) 172225 (1.29%) 596202 (6.74%) 178337 (2.76%) 2058443 (13.40%) 17067 (0.34%) 51101 (0.69%) 49227 (0.74%) 121413 (2.48%)
Total bps coverred, bp 2146219 2001793 797791 835527 914841 767267 1282002 738538
Genome coverage 46.26% 43.15% 17.20% 18.01% 19.72% 16.54% 27.63% 15.92%


Sequence Read Name s_4_pe_ID1_NX s_4_pe_ID2_NX s_4_pe_ID3_NX s_4_pe_ID4_NX s_4_pe_ID5_NX s_4_pe_ID6_NX s_4_pe_ID7_NX s_4_pe_ID8_NX
1000000 uniqe reads 4948046 1542020 3244646 1792757 6835270 2481390 6341154 3074085
700000 uniqe reads 4784689 1736973
Proccessed reads 4948046 1542020 3244646 1792757 4784689 1736973 6341154 3074085
Mapped read, no. (unique) 1028262 (20.78%) 1047304 (67.92%) 1032729 (31.83%) 1060752 (59.17%) 701069 (14.65%) 721899 (41.56%) 1001553 (15.79%) 1029838 (33.50%)
Failed to align, no. 3853931 (77.89%) 381914 (24.77%) 2118408 (65.29%) 467033 (26.05%) 4067507 (85.01%) 1002826 (57.73%) 5292536 (83.46%) 1965747 (63.95%)
Mapped read, no (non-unique) 65853 (1.33%) 112802 (7.32%) 93509 (2.88%) 264972 (14.78%) 16113 (0.34%) 12248 (0.71%) 47065 (0.74%) 78500 (2.55%)
Total bps coverred, bp 1890835 1445489 652120 310201 897507 456971 1267625 676415
Genome coverage 40.76% 31.16% 14.06% 6.69% 19.35% 9.85% 27.32% 14.58%

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.