CpG count query

[MarkOEze]

Hello,

I have some DNA methylation data, which I prepared using EM-Seq and got around 236 million paired-end reads after sequencing on the Illumina NextSeq platform. I have some concerns regarding the number of CpGs I obtained. It is known that there are 28 million CpGs in the human genome; however, the number of CpGs I got, especially at 1X, 3X, and 5x coverage, suggests that the CpG counts may have been overestimated.

I trimmed my reads using Trimgalore, aligned them and extracted the methylation information using Bismark. Please look at the snippets of my codes and the corresponding results below.

Alignment: bismark --bowtie2 --gzip -N 1 --un --ambiguous --phred33-quals -p 4 --multicore 20 --genome /mnt/hcs/dsm-pathology-chatterjee-genomics/Euan_Rodger/Ref_Genomes/GRCh37 -1 AAFJNNMM5-8861-04-00-01_S1_L001_R1_001_val_1.fq.gz -2 AAFJNNMM5-8861-04-00-01_S1_L001_R2_001_val_2.fq.gz

Methylation extraction: bismark_methylation_extractor --gzip --bedGraph --comprehensive --s --multicore 10 --merge_non_CpG --cutoff 1 -o ./bedgraph_10_2/ AAFJNNMM5-8861-06-00-01_S3_L001_R1_001_val_1_bismark_bt2_pe.bam

N/B: I repeated this for all the samples at 3X, 5X, and 10X coverage (codes not shown).

Sample	1x coverage	3x coverage	5x coverage	10x coverage
S1	48,841,280	29,043,575	12,581,307	650,604
S2	50,761,706	34,860,543	18,166,369	1,472,475
S3	44,959,784	21,303,364	7,247,631	265,220

I also counted the number of CpGs with methylkit using the bam files obtained from the Bismark alignment. Result:

Sample	1x coverage	3x coverage	5x coverage	10x coverage
S1	49,783,470	31,603,367	16,688,990	1,992,364
S2	52,039,475	37,017,454	22,484,852	3,749,399
S3	45,501,338	26,431,339	13,237,981	1,623,263

Although the second approach seems to give more reasonable results, I still have serious concerns about the accuracy of the CpG count at 1x, 3x, and 5x coverage.

Sorry about the long text; I wanted to provide as much relevant information as possible. Please let me know if you require more information.

I look forward to hearing from you soon.

Thank you.

Kind regards, Mark.