Question: Conversion effinciency issue in whole genome bisulfite sequencing data.
0
gravatar for Tori
3.5 years ago by
Tori80
Finland
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 3.5 years ago by Giovanni M Dall'Olio26k • written 3.5 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 3.5 years ago by Devon Ryan90k
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