Sam:LabNotes/Microbiome-new/2011-2-15: Difference between revisions
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{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''Sequence Read Name''' | | align="center" style="background:#f0f0f0;"|'''Sequence Read Name''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''s_4_pe_ID1_NX''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''s_4_pe_ID2_NX''' | ||
| align="center" style="background:#f0f0f0;"|''' | | 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_ID4_NX''' | ||
| align="center" style="background:#f0f0f0;"|'''s_4_pe_ID5_NX''' | | align="center" style="background:#f0f0f0;"|'''s_4_pe_ID5_NX''' |
Revision as of 01:04, 28 February 2011
Bowtie mapping(whole reads) and contamination analysis(failed to aligned reads)- HC-MDA-Ecoli-SAGs(NX1-NX8)-Jan09
Background
- Ecoli SAGs were prepared from flow-sorted single E.coli cells.
- Eight E.coli SAGs samples were made to Nextera-libraries.
- 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.
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% |
- 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.