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

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>Sam Chiang
Line 42: Line 42:
**Bowtie setting: -k 1 -l 28 -n 2 -m 1 --best --best --phred64-quals
**Bowtie setting: -k 1 -l 28 -n 2 -m 1 --best --best --phred64-quals
**Downstream: Data was used for genome coverage calculation.
**Downstream: Data was used for genome coverage calculation.
  [[File:021511-bowtie-mapping-Ecoli-SAG-NX.png|800px]]
 
result
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sequence Read Name'''
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID1_NX'''
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID2_NX'''
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID3_NX'''
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID4_NX'''
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID5_NX'''
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID6_NX'''
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID7_NX'''
| align="center" style="background:#f0f0f0;"|'''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%
|-
|
|}
 
 
 
  [[File:021511-bowtie-mapping-Ecoli-SAG-NX.png|1300px]]





Revision as of 00:56, 28 February 2011

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 &


General genome coverage analysis

  • Whole reads alignment using Bowtie => genome coverage calculate
    • Script: Read2coverage-kz-3
    • Data resource: pooled pe reads data from HC-MDA-Ecoli-SAGs(NX1-NX8)-Jan09
    • 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.

result

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%


File:021511-bowtie-mapping-Ecoli-SAG-NX.png







  • 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.