Sam:LabNotes/Microbiome-new/2011-4-14

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Revision as of 08:51, 18 April 2011 by >Sam Chiang (→‎Results)
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Quantify low quality E.coli reads from Ecoli SAGs sequencing results

Background

  • Since bowtie mapping is very stringent, some E.coli reads (assuming they are low-quality E.coli reads) are not able to be mapped.
  • Total reads = mappled E.coli reads + non-mapped E.coli reads + non-mapped non-E.coli reads
    • The objective here is to quantify how many of those non-mapped E.coli reads.
  • We can't not directly BLASTN all of the failed-to-align reads since it will take extremely long time (> 1 week) using genemapster.

Possible approaches

Test1: BLATing all failed-to-align reads

  • BLAT failed-to-align reads to E.coli genome -> output in BLAST-output format -> MEGAN extraction of E.coli reads and get quantitative information.
  • Since the failed-to-aling reads (e.g. s_4_ID1.failed) are FASTQ file, I need to trasform them to FASTA files before I can BLAT them.
    • Using script fastq2fasta-hc.pl to transfer FASTQ format to FASTA format
BLAT command:
 
samchiang@genemapster:~/Tools/blatSuite34_64$ ./blat -out=blast /media/disk-1/samchiang/Raw-Read-Complete/101123_HL083/HC-MDA-Ecoli-SAGs-Ind1to8-SE/failed-or-nonspecific/s_4_ID1.failed.fa /media/disk-2/samchiang/Tools/bowtie-0.12.5/genomes/e_coli_k12_mg1655.fasta /media/disk-1/samchiang/Sub_tests_folder_imp/3_Quantify_lowquality_reads_Ecoli_SAGs/HL083_Ecoli_SAG_ID1.failed.fa.BLAT.txt &
  • Results
    • It is unclear why but The BLAT result (in BLAST output format) doesn't contain any header-string information. So MEGAN can't identify taxonomic information from BLAT results.
    • I did several tests and confirm that it's not due to the output format (blast or .psl) I chose. It's probably due to that the reference genome is a one string FASTA file.

Test2: bowtie mapping to gigantic reference genome

  • Whole genome de novo assembly by ABYSS -> BLASTN contigs -> MEGAN extraction of E.coli contigs -> Joint E.coli contigs with E.coli reference genome -> build up gigantic reference genome -> map the raw reads to gigantic reference genome to count the failed-to-align reads
  • Started from whole genome ABYSS-pe assembly 100up data (by Chris)
    • ID1_Ecoli.abyss.pe.K27.contigs.100up ~ ID8_Ecoli.abyss.pe.K27.contigs.100up
  • BLASTN the contigs and extract E.coli contigs by MEGAN
  • Cat the E.coli reference genome with the MEGAN extracted E.coli contigs
samchiang@genemapster:/media/disk-1/samchiang/Sub_tests_folder_imp/3_Quantify_lowquality_reads_Ecoli_SAGs/Step2_Megan_output/MEGAN output$ cat e_coli_k12_mg1655.fasta ID8_Ecoli_contigs_100up_including_summarized.new > Ecoli_gigentic_ID8 &
  • Build up bowtie gigantic E.coli genome
samchiang@genemapster:~/Tools/bowtie-0.12.5$ ./bowtie-build /media/disk-1/samchiang/Sub_tests_folder_imp/3_Quantify_lowquality_reads_Ecoli_SAGs/Step2_Megan_output/MEGAN_output/Ecoli_gigentic_ID2 indexes/Ecoli_gigentic_index_ID2/Ecoli_gigentic_index_ID2 &
  • Map the raw reads to Ecoli_gigantic_index
samchiang@genemapster:~/Scripts$ perl bowtie_gigantic_ID1_map /media/disk-1/samchiang/Raw-Read-Complete/101123_HL083/HC-MDA-Ecoli-SAGs-Ind1to8-PE/s_4_1and2_ID1.txt /media/disk-1/samchiang/Raw-Read-Complete/101123_HL083/HC-MDA-Ecoli-SAGs-Ind1to8-PE/s_4_1and2_ID1.failed.gig /media/disk-1/samchiang/Raw-Read-Complete/101123_HL083/HC-MDA-Ecoli-SAGs-Ind1to8-PE/s_4_1and2_ID1.nonunique.gig &

Results

File:04142011-quantifying low quality Ecoli reads-new.png

Follow up

  • I did another similar test to make gigantic reference using E.coli reference + E.coli contigs (assembled from raw reads) + E.coli contigs (assembled from failed-to-align reads), the result wasn't improved either.