Can it be used for amplification based targeted methylation analysis?

[vrbacky]

Hi,

gemBS seems to be a very interesting toolkit. My wet lab colleagues use targeted approach - they amplify specific regions of bisulfite converted DNA and analyze it using amplicon sequening on Illumina MiSEQ. I came to their data rather by an accident because they were unable to use their methods.

I've tried to analyze their data using gemBS. I can map the data without any problem - paired end data are mapped to a sequence used as a reference. Almost all reads are mapped and are reported as correct pairs in gemBS mapping report. Samtools flagstat reports them as properly paired too. But I get a lot of warning and errors during methylation and variant calling. All reads are reported as follows in calling err file:

[W::vcf_parse] Contig 'Warning not found: M03647:172:000000000-D4RY5:1:1102:4243:8025 4858 4863 +' is not defined in the header. (Quick workaround: index the file with tabix.)
...
[E::bcf_write] Broken VCF record, the number of columns at Warning not found: M03647:172:000000000-D4RY5:1:1102:4243:8025 4858 4863 +:1 does not match the number of samples (0 vs 1)
...

I can avoid the messages by changing keep_improper_pairs option to False but I get all reads as PairNotFound in calling json log.

Is there any way how to analyze such data?

Thank you very much.

My config is:

# Required section
#
# Note that the index and contig_sizes files are generated from the
# reference file if they do not already exist
#

reference = reference/ctnnd2.fa

#
# This is for the control sequences.  The contigs here will
# be used for mapping, but will not be passed to the caller
#
# extra_references = reference/conversion_control.fa.gz

index_dir = indexes

#
# The variables below define the directory structure for the results files
# This structure should not be changed after the analysis has started
#

base = .
sequence_dir = ${base}/fastq
bam_dir = ${base}/mapping/@BARCODE
bcf_dir = ${base}/calls/@BARCODE
extract_dir = ${base}/extract/@BARCODE
report_dir = ${base}/report

#
# End of required section
#


# The following are optional

project = gembs_test_ctnnd2
species = human

threads = 20
jobs = 6

[index]

sampling_rate = 4

[mapping]

non_stranded = False
remove_individual_bams = True

#underconversion_sequence = NC_001416.1 
#overconversion_sequence = NC_001604.1

[calling]

mapq_threshold = 10
qual_threshold = 13
reference_bias = 2
left_trim = 5
right_trim = 0
keep_improper_pairs = True
keep_duplicates = True
haploid = False
conversion = auto
remove_individual_bcfs = True

# Contigs smaller than contig_pool_limit will be called together
contig_pool_limit = 25000000

[extract]

strand_specific = True
phred_threshold = 10
make_cpg = True
make_non_cpg = True
make_bedmethyl = True
make_bigwig = True

[heathsc]

Thanks for the bug report. The original error was caused by a warning message that was finding its way into the vcf output causing problems downstream. I have fixed this, but the problem remains that for some reason bscall is having problems finding both members of a pair. Is there any chance that you could share a part of your input bam file so that I can see what is happening?

[heathsc]

You can send the file directly to my email address to avoid sharing it with everyone... ([email protected])

[vrbacky]

Sure. Thank you very much. More by email.