Sam:LabNotes/Microbiome-new/2011-4-14: Difference between revisions

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**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.
**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 gigentic reference genome===
===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 refrence genome -> build up gigentic reference genome -> map the raw reads to gigentic reference genome to count the failed-to-align reads
*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

Revision as of 08:20, 18 April 2011

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