[mira_talk] Alignment errors in Solexa mapping
- From: Björn Nystedt <bjorn.nystedt@xxxxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Thu, 10 Dec 2009 13:34:42 +0100
Hi all,
having fun learning to work with Solexa reads :)
I was mapping Solexa reads to a reference genome (= a single contig from an
earlier MIRA assembly) using MIRA V3rc4d (sorry Bastien, missed the strain info
again..)
mira --project=BAnh1IrMREF07 --job=mapping,genome,accurate,solexa -OUT:ora=yes
-GE:not=3 -AS:
urd=yes -SB:lb=yes:bft=fasta:bbq=30 SOLEXA_SETTINGS -LR:ft=fastq:fqqo=64
-CO:msr=yes -GE:uti=
no:tismin=200:tismax=400
Going through the alignments, I noticed some odd parts, such as this:
attggcgtgaagagtttgaatgagattgattgmmwgccagagaggccagaatatgttacaccgccgtcga
(consensus)
attggcgtgaagagtttgaatgagattgattgcmagccagagaggccagaatatgttacaccgccgtcga
(reference/backbone)
attggcgtgaagagtttgaatgagattgattgccag**agn
attggcgtgaagagtttgaatgagattgattgccag**agn
attggcgtgaagagtttgaatgagattgattgccag**agn
ttggcgtgaagagtttgaatgagattgattgccag**agan
tggcgtgaagagtttgaatgagattgattgccag**agagn
ggcgtgaagagtttgaatgagattgattgccag**agag*gn
ggcgtgaagagtttgaatgagattgattgccag**agag*gn
ggcgtgaagagtttgaatgagattgattgccag**agag*gn
gtgaagagtttgaatgagattgatt****gccagagaggccan
gtgaagagtttgaatgagattgatt****gccagagaggccan
gtgaagagtttgaatgagattgatt****gccagagaggccan
gtgaagagtttgaatgagattgatt****gccagagaggccan
tgaagagtttgaatgagattgatt****gccagagaggccagn
tgaagagtttgaatgagattgatt****gccagagaggccagn
gattgattgccagagaggccagaatatgttacaccgccn
attgccagagaggccagaatatgttacaccgccgtcga
(Note that these are only a few selected reads in the alignment; we have ~120X
at this site which is normal given the amount of data). As you see, there are 3
predicted variants (all three are present in both strand directions):
1) ttgccag**agag*g (30 reads)
2) tt****gccagagag (70 reads)
3) ttgattgccagagag (20 reads)
This is an overprediction of the diversity, since the two top reads are really
identical.
Anyone seen this behaviour before?
Could it be caused by the wildcard charachers in the reference (although
similar things sometimes happens also when there are no wildcards)?
Any ideas on strategies/parameters to avoid it?
A similar error is quite common (mostly at the end of reads), at sites where
there are (homopolymer) frameshifts in the reference genome:
aaagacgataaaaaatatcgcaatgaaaaataaaaaagawtttttttgttttatc(consensus)
aaagacgataaaaaatatcgcaatgaaaaat*aaaaagaatttttttgttttatcac (reference/backbone)
nataaaaaatatcgcaatgaaaaataaaaaag*atttttt
naaaaaatatcgcaatgaaaaataaaaaagatttttttt
Again, MIRA overpredicts diversity, since these two reads are identical, and
there should be no a/t ambiguity (w) in the consensus in this case.
Any help or comments are welcome!
B
PS. In our case the problems below can likely be avoided by doing a de-novo or
de-novo-like assembly with all our current data, but we are working on a range
of strategies at the moment to learn about different possibilities, and I
thought the aboce behaviours were of general interest and worth discussing on
the list.
PS2. I am a bit confused regarding the initial n:s of the reads, not sure yet
why they appear...
====================================
Björn Nystedt, PhD
Molecular Evolution
EBC, Uppsala University
Norbyv. 18C, 752 36 Uppsala
Sweden
phone: +46 (0)18-471 45 88
email: Bjorn.Nystedt@xxxxxxxxx
====================================
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