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='''General statiscs (whole reads) and contamination analysis (failed-to-align reads) - HC-MDA-Ecoli-SAGs-Ind1to8'''= ==Background== *Samples were prepared from -(short and long amplified E.coli SAGs -3rd test 11-07-'10) *Eight E.coli SAGs samples were made to Illumina sequencing libraries using (N2)Primer-based barcodes ID1 to ID8. *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. *Since I don't have genome-tech server permission, I asked Dinh's help to transfer data into Genemapster server. ==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 (SE reads data set) **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|800px]] *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|700px]] *Length filtering and keep only >300bp contigs. **Script: seqLenFilger.pl *Perform BLAST on filtered contigs **BLAST setting: -K 1 -m 8 -a 2 -v 1 βb 1(using -m 8 output format to extract GI number) **BLAST setting: -K 1 -m 8 -a 2 -v 1 βb 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 Batch Entrez (against nucletide database). Transform GI number to UID for taxonomic analysis ===Results=== [[File:sam120810-failed to aligned ID2-4.jpg|px300]] [[File:sam120810-failed to aligned ID5-8.jpg|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'''. γ 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. ==Testing using less-stringent report suppression setting on bowtie mapping== *I noticed that the genome coverage calculation script (Read2coverage-kz-3) calculate the coverage from bowtie.out file, however the bowtie.out file only report unique mapped reads (confirmed on http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Microbiome-new/2010-12-13). **I guess there are genome coverages not counted if they are from non-unique mapped reads, since non-uniqe mapped reads were not reported in bowtie.out. ===Re-analyze genome coverage using new scripts with less-stringent report suppression=== *test data set: s_4_ID6.txt from SE data set (HL083 E.coli SAGs) **This data set has the highest genome coverage than others. *'''Read2coverage-kz-3-suppress_5''' **(allow reporting 5 alignments from 1 read) *Results # reads processed: 2372473 # reads with at least one reported alignment: 1512449 (63.75%) # reads that failed to align: 512805 (21.61%) # reads with alignments suppressed due to -m: 347219 (14.64%) Reported 1512449 alignments to 1 output stream(s) total bps coverred = 1824363 *'''Read2coverage-kz-3-suppress_10''' **(allow reporting 10 alignments from 1 read) *Results # reads processed: 2372473 # reads with at least one reported alignment: 1857421 (78.29%) # reads that failed to align: 512805 (21.61%) # reads with alignments suppressed due to -m: 2247 (0.09%) Reported 1857421 alignments to 1 output stream(s) total bps coverred = 1869404 ==Conclusions== *Test data: E.coli SAG HL083(SE) s_4_ID6.txt {| {{table}} | align="center" style="background:#f0f0f0;"|'''Report suppressor''' | align="center" style="background:#f0f0f0;"|'''m = 1''' | align="center" style="background:#f0f0f0;"|'''m = 5''' | align="center" style="background:#f0f0f0;"|''' m= 10''' |- | proccessed reads||2372473||2372473||2372473 |- | unique alignment||1411597 (59.50%)||1512449 (63.75%)||1857421 (78.29%) |- | failed-to-align||512805 (21.61%)||512805 (21.61%)||512805 (21.61%) |- | non-unique alignment||448071 (18.89%)||347219 (14.64%)||2247 (0.09%) |- | Total covered bp||1778296||1824363||1869404 |- | Coverage (E.coli K12)||38.33%||39.32%||40.30% |- | |} *Tune down the report suppression strigency did work on increasing genome coverage, but the effect is limited. Even with m=10 setting where alomst all non-unique alignments (18%) were reported (counted in genome coverage), the genome coverage was only increased by ~2% at most. *'''The genome coverage is mainly contributed by unique alignment'''. ==Identify where did those non-unique reads map== *Perform ABYSS on non-unique read of s_4_ID6.nonspecific reads *I mapped the assembled contigs on E.coli reference genome using BLAT, and found that these contigs are randomly distributed.γ
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