Question: Question about RRBS mapping efficiency
0
gravatar for hxlei613
3.3 years ago by
hxlei61390
Bangladesh
hxlei61390 wrote:

I've been working on RRBS data these days. Raw data were processed using Trim_galore tool, in order to remove adapter and low-quality reads. The clean data were mapped to the reference genome using the Bismark alignment tool with default parameters(--bowtie2 --directional). Final Alignment report is like this:

Sequence pairs analysed in total:       17109021
Number of paired-end alignments with a unique best hit: 10941462
Mapping efficiency:     64.0%
Sequence pairs with no alignments under any condition:  3219358
Sequence pairs did not map uniquely:    2948201
Sequence pairs which were discarded because genomic sequence could not be extracted:    0

Total number of C's analysed:   539400501

Total methylated C's in CpG context:    28032910
Total methylated C's in CHG context:    2657563
Total methylated C's in CHH context:    5817321
Total methylated C's in Unknown context:        0

Total unmethylated C's in CpG context:  90723174
Total unmethylated C's in CHG context:  147210060
Total unmethylated C's in CHH context:  264959473
Total unmethylated C's in Unknown context:      1

C methylated in CpG context:    23.6%
C methylated in CHG context:    1.8%
C methylated in CHH context:    2.1%
C methylated in unknown context (CN or CHN):    0.0%

Is the resualt ok ? I want to know wether the 64% mapping efficiency is ok for RRBS data and why. And why is it so low compared to other NGS ?

Thank you very much.

rrbs mapping efficiency • 2.1k views
ADD COMMENTlink modified 3.3 years ago by Devon Ryan92k • written 3.3 years ago by hxlei61390
2
gravatar for Devon Ryan
3.3 years ago by
Devon Ryan92k
Freiburg, Germany
Devon Ryan92k wrote:

The methylation percentages seem pretty reasonable (normally you expect CHG and CHH methylation to be 1 or 2 percent). Not knowing what species this is, it's hard to judge if 64% alignment is good or now. For mouse I normally got higher (80-90%), but either way it's not terrible and with RRBS you probably have enough coverage as is.

BTW, RRBS has a lower alignment rate than other things because you only have 3 nucleotides to work with when doing the alignment. Consequently, there are a lot of reads that map to a million places and are consequently just tossed.

ADD COMMENTlink written 3.3 years ago by Devon Ryan92k

I forgot to say ... it's human.

ADD REPLYlink written 3.3 years ago by hxlei61390

That seems a bit of a low alignment rate to me then. What happens if you instead use bwa-meth? Just try that on one sample and see what sort of alignment percentage you get out.

ADD REPLYlink written 3.3 years ago by Devon Ryan92k

Ok,I will try. By the way, what is the common RRBS alignment rate for human ?

ADD REPLYlink written 3.3 years ago by hxlei61390

I would expect 80-90%.

ADD REPLYlink written 3.3 years ago by Devon Ryan92k

Devon, just a follow up on this thread. Is a mapping efficiency between 35% and 45% too low for bovine?

ADD REPLYlink written 2.7 years ago by mrodrigues.fernanda40
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