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
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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
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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
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?