Question: Conversion effinciency issue in whole genome bisulfite sequencing data.
gravatar for Tori
3.2 years ago by
Tori80 wrote:

I have samples from Whole Genome Bisulfite Sequencing. Lambda genome has been spiked-in. I calculate bisulfite conversion efficiency by mapping the data to lambda genome and calculate conversion efficiency using following equation.

Conversion efficiency = 100 - {no. of methylated Cs in CpG context/(no of methylated Cs in CpG context + no of unmethylated Cs in CpG context)} %

For one of the samples I got strange statistics. There were very few number of C analysed compared to other samples and all Cs were methylated. By using the numbers given by Bismark  I got 0% conversion efficiency. What could have gone wrong? Has the bisulfite conversion failed or could there be any other reasons? When I mapped the data to reference genome I got 70% mapping efficiency. Following is the mapping summary from Bismark (mapped to lambda genome).


Final Alignment report
Sequences analysed in total:    3973593
Number of alignments with a unique best hit from the different alignments:    1
Mapping efficiency:    0.0%
Sequences with no alignments under any condition:    3973592
Sequences did not map uniquely:    0
Sequences which were discarded because genomic sequence could not be extracted:    0

Number of sequences with unique best (first) alignment came from the bowtie output:
CT/CT:    1    ((converted) top strand)
CT/GA:    0    ((converted) bottom strand)
GA/CT:    0    (complementary to (converted) top strand)
GA/GA:    0    (complementary to (converted) bottom strand)

Number of alignments to (merely theoretical) complementary strands being rejected in total:    0

Final Cytosine Methylation Report
Total number of C's analysed:    13

Total methylated C's in CpG context:    2
Total methylated C's in CHG context:    3
Total methylated C's in CHH context:    8
Total methylated C's in Unknown context:    0

Total unmethylated C's in CpG context:    0
Total unmethylated C's in CHG context:    0
Total unmethylated C's in CHH context:    0
Total unmethylated C's in Unknown context:    0

C methylated in CpG context:    100.0%
C methylated in CHG context:    100.0%
C methylated in CHH context:    100.0%
Can't determine percentage of methylated Cs in Unknown context (CN or CHN) if value was 0


ADD COMMENTlink modified 3.2 years ago by Giovanni M Dall'Olio26k • written 3.2 years ago by Tori80

You only got one read aligned, so the other metrics are meaningless. Could it be that the reads for the spiked-in lambda have already been removed?

ADD REPLYlink written 3.2 years ago by Devon Ryan88k
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 2480 users visited in the last hour