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
- 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
$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