Sam:LabNotes/Microbiome-new/2011-2-15: Difference between revisions
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==Background== | ==Background== | ||
* | *Ecoli SAGs were prepared from flow-sorted single E.coli cells. | ||
*Eight E.coli SAGs samples were made to | *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. | ||
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
- 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
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.