Sam:LabNotes/Microbiome-new/2011-2-15
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Bowtie mapping on E.coli SAG Nextera libraries HC-MDA-Ecoli-SAGs(NX1-NX8)-Jan09[edit]
Background[edit]
- 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[edit]
Data pooling[edit]
- 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)[edit]
- 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
Results[edit]
- Original excel table Media:021511_whole-read-bowtie-NX1-8.xls
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% |
Discussions[edit]
Mappalbe rates[edit]
- The mappalbe rates are very low in these Nextera libraries (NX_ID_1 ~ NX_ID8), even worse then previous standard Illumina libraries (E.coli SAG) http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-12-8.
- The low mappable rates are due to high failed-to-align rates, which could be caused by:
- 1. Exogeneous DNA contamination -> Validate failed-to-align reads (or de novo assembled contigs) by BLASTn or MEGAN.
- 2. Low quality of reads or containing sequencing tail -> Manually check the read structure
If it is true that exogeneous DNA contamination is the major issue of libraries quality, then using Nexteral-libraries construction were not albe to reduce DNA contamination effectively. It is possible that the primary exogeneous DNA contamination came from prior MDA amplificaiont, so changing library construction won't help much.
Genome coverage[edit]
- Genome coverage were compared under the same number of unique reads (down-sampled to 1,000,000 unique reads).
- Libraries NX-ID5 and NX-ID6 were down-sampled to 700,000 unique reads due to the insufficient available mappalbe reads.
- In the short-vs-long paired comparisons (NX-ID1 vs NX-ID2, NX-ID3 vs NX-ID4, NX-ID5 vs NX-ID6, NX-ID7 vs NX-ID8), short-amplified libraries always showed higher (1.5~2 fold) genome coverage than long-amplified libraries.