Dinh/Dinh 2013/NOTES/2013-10-7: Difference between revisions

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= MEGAN analysis of unmapped reads=
= Blueprint Capture, low mapping issue =
* Low mapping can be caused by any of the following problems
# Contamination - generally, contamination would not be amplified by the last PCR step or sequenced due to lack of adapter sequences
# Chimeric capture sequences - when imperfect hybridization of capture arms to genomic sequences which occurs frequently in the genome, leading to off-target & chimeric sequences. However, when we remove the capture arms, we should get recover the alignments for these regions, so chimeric capture sequences may not be the case.
# Error from sequencing - Sequencing quality is good from HiSeq Rapid Run.
# Error from oligo synthesis - error from oligo synthesis would still allow us to recover the alignments from the off-target regions, & even so, should lower the specificity of capture, but this is not the case.
== MEGAN analysis of unmapped reads==


* I mapped the first 1000 unmappable reads with blastn
* I mapped the first 1000 unmappable reads with blastn

Revision as of 23:57, 7 October 2013

Blueprint Capture, low mapping issue

  • Low mapping can be caused by any of the following problems
  1. Contamination - generally, contamination would not be amplified by the last PCR step or sequenced due to lack of adapter sequences
  2. Chimeric capture sequences - when imperfect hybridization of capture arms to genomic sequences which occurs frequently in the genome, leading to off-target & chimeric sequences. However, when we remove the capture arms, we should get recover the alignments for these regions, so chimeric capture sequences may not be the case.
  3. Error from sequencing - Sequencing quality is good from HiSeq Rapid Run.
  4. Error from oligo synthesis - error from oligo synthesis would still allow us to recover the alignments from the off-target regions, & even so, should lower the specificity of capture, but this is not the case.

MEGAN analysis of unmapped reads

  • I mapped the first 1000 unmappable reads with blastn
 $blastn -p blastn -d nt -K 100 -e 100 -g F -F "m D" -a 16 -W 7 -i Indx12.fa -o Indx12.bwa.R1.blast
  • Result from MEGAN (Indx12, Indx13, Indx14 only):
 File:Indx12.bwa.R1.megan.png
 File:Indx13.bwa.R1.megan.png
 File:Indx14.bwa.R1.megan.png