About modification caller

[weishwu]

I read in your paper that Nanopolish performed the best among the 3 callers you tested. I have a few questions:

1. Nanopolish clusters neighboring CpGs when they are very close to each other. In the outputs I got from Nanopolish, ~15% methylation calls are for a cluster of 2-50 CpGs. Is this a problem? Should I assume these CpGs are less accurate and only use the CpGs that got measured independently? When I asked this under Nanopolish git, they said this limitation was due to the training data they used which was 100% methylated. They recommended me to use dorado if I need absolute single-CpG methylation.

2. Megalodon is another caller compatible with NanoMethPhase. However it's deprecated by Nanopore and is replaced by dorado (https://github.com/nanoporetech/dorado). Is NanoMethPhase compatible with dorado outputs?

3. About deepsignal, the team has developed deepsignal2 which has better performance in 5mCpG detection of human. Is deepsignal2 compatible with NanoMethphase?

Thanks.

[vahidAK]

Hi @weishwu,

I suggest keeping clustered CpGs. NanoMethPhase splits clustered CpGs to per CpG so it should not be a problem.

For other methylation callers, as long as the output of your methylation caller is what I have explained at the end of the README file it should be fine for NanoMethPhase. However, because nanomethphase matches the read coordinates from methylcall file to the read coordinates from the bam file, the optimal results will be when the same bam or alignments used for methylation calling are used for NanoMethPhase phasing.

Best, Vahid

[assafgrw]

Hi, Regarding mod basecalling, I am afraid that Nanopolish is not compatible with the new ONT chemistry (V14). Do you think that Doardo will do the job?

[vahidAK]

It should be able to call methylation for v14. Guppy with the appropriate model can also be used to store methylation in a bam file.

[weishwu]

Hi @vahidAK. I've run mod base calling using Dorado. Dorado itself only outputs a BAM file with MM/ML tags. I had to run modkit extract to extract the modification information. The format of the output is like below. It has only one probability score (mod_qual) while all the 3 methylation callers compatible with NanoMethPhase have two scores. Is there a way to transform this to make it compatible with NanoMethPhase?

read_id forward_read_position   ref_position    chrom   mod_strand  ref_strand  ref_mod_stranfw_soft_clipped_start  fw_soft_clipped_end read_length mod_qual    mod_code    base_qual   ref_kmer    query_kmer  canonical_base  modified_primary_base   inferred
0a0a9f4b-6e9f-4e99-9e9e-c639c35f39c2    557 17158282    chr9    +   -   -   0   794 0.013671875 h   9   CCGAA   TTCGG   C   C   false
0a0a9f4b-6e9f-4e99-9e9e-c639c35f39c2    557 17158282    chr9    +   -   -   0   794 0.017578125 m   9   CCGAA   TTCGG   C   C   false
0a0a9f4b-6e9f-4e99-9e9e-c639c35f39c2    103 17158737    chr9    +   -   -   0   794 0.013671875 h   13  TCGTA   TACGA   C   C   false

[vahidAK]

Hi @weishwu I guess using something like :

awk -F'\t' '$11=="m" && $3>=0 {print $4,$3,$6,"NA",$1,"NA",1-$10,$10,"NA",$13}' OFS='\t'

you should be able to convert this to a DeepSignal-like format and then use nanomethphase. Alternatively, you should be able to haplotag your bam file and use modkit to extract H1 and H2 methylation frequencies from the bam file.

[weishwu]

@vahidAK Thanks! Yes, I went through the alternative way by splitting the bam into h1 and h2, running modkit pileup, and then DSS. Another question: do you think it makes sense to use only the CpGs covered by at least 5 reads (cov >= 5) for DSS test? That's the cutoff I usually use for Illumina WGBS data. Not sure if it's still a reasonable filter for ONT.

[vahidAK]

I suggest not to filter because DSS takes care of this and depth will be factored into the statistical computation. See here for more info.

[weishwu]

@vahidAK . Got it. Thanks!

[weishwu]

Hi @vahidAK Sorry to reopen this. I reformatted the modkit output according to what you told me above, and then ran methyl_call_processor. After that, phase gave me an error.

The top lines of the input to methyl_call_processor:

chrom   pos strand  pos_in_strand   readname    read_strand prob_0  prob_1  called_label    k_mer
chr1    10468   +   NA  8f53107b-f511-4363-8c04-bc3424d54b40    NA  0.994141    0.005859375 NA  CTCGC
chr1    10470   +   NA  8f53107b-f511-4363-8c04-bc3424d54b40    NA  0.998047    0.001953125 NA  CGCGG
chr1    10484   +   NA  8f53107b-f511-4363-8c04-bc3424d54b40    NA  0.990234    0.009765625 NA  CCGGC
chr1    10488   +   NA  8f53107b-f511-4363-8c04-bc3424d54b40    NA  0.994141    0.005859375 NA  CCCGC

My command-lines are:

# methyl_call_processor
python /usr/share/NanoMethPhase/nanomethphase.py methyl_call_processor --tool_and_callthresh deepsignal:0.6 -mc {input}.reformatted.tsv -t {threads} | sort -k1,1 -k2,2n -k3,3n | bgzip > {output.bedgz}
tabix -p bed {output.bedgz}

# phase
python /usr/share/NanoMethPhase/nanomethphase.py phase --include_indels -b {input.aln} -v {input.vcf} -mc {input.meth} -r {params.genome_fasta} -o {params.out_prefix} -of bam,methylcall,bam2bis -t {threads}

The full log file is attached. The tail of the log is:

Tagging variants to reads from chr9: 100%|██████████ [ Estimated time left: 00:00 ]
Tagging variants to reads from chrX: 100%|██████████ [ Estimated time left: 00:00 ]
Tagging variants to reads from chrY: 100%|██████████ [ Estimated time left: 00:00 ]
Read Seperation Process Started
Processing reads from chr1:   1%|          | 14900/1292811 [00:14<21:07, 1008.50it/s] 
Traceback (most recent call last):
  File "/usr/share/NanoMethPhase/nanomethphase.py", line 2242, in <module>
    main()
  File "/usr/share/NanoMethPhase/nanomethphase.py", line 2236, in main
    args.func(args)
  File "/usr/share/NanoMethPhase/nanomethphase.py", line 1194, in main_phase
    alignmentwriter(result, outHP22BisSam)
  File "/usr/share/NanoMethPhase/nanomethphase.py", line 411, in alignmentwriter
    out_samRead.set_tags(all_tags)
  File "pysam/libcalignedsegment.pyx", line 2656, in pysam.libcalignedsegment.AlignedSegment.set_tags
  File "pysam/libcalignedsegment.pyx", line 385, in pysam.libcalignedsegment.pack_tags
AttributeError: module 'array' has no attribute 'typecode'

The alignment was generated using dorado aligner and then coordinate-sorted. Variants were called using Clair3 and then phased using Whatshap.

Below is the first line in the BAM. My guess is that some of the tags cannot be processed by pysam?

0a8e9d66-3338-4578-a7ff-fbe08d4e2434    2048    chr1    10001   23  169S15M1D32M1D20M1D11M1D34M1D29M1D30M7I57M1D18M2D5M1D63M12D3M1D13M4D32M1D13M1I18M1I6M1I15M3602S *   0   0   CCCTAACCCTAACCCTCACCCCTAACCCCTAACCCTAACCCTAACCCTGACCCCTAACCCTAACCCTGACCCTAACCCTAACCCTAACCCTAACCCTCACCCTGACCCTCACCCTGACCCTGACCCTGACCCATAACCCATAACCCTAACCCTAACCCTAACCCTAACCTAACCCTAACCCTAACCTAACCCTAACCCTAACCCTAACCCTAACCCAACCCTAACCCTAACCCTAACCTAACCCTAACCTAACCCTAACCCTAACCCTAACCTAACCCTAACCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCCGACCCTGACCCTAACCCTAACCCTAACCCTAACCCTGACCCTGACCCTAACCCTAACCCCAACCCCAACCCCAACCCCGGCCCAACCCCAACCCTAACCCTAACCCTAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCCAACCCTAACCCTAAACCCTAACCCTAACCCTAACCCTAACCCCAACCCTAACCCCTAACCCCTAACCCTAACCCTAACCCCTAACCCCTAACCCTAACCCAAATAATCACTCAAAATCATCCTTACACTAAAAATGCTAAACTATACAATTTCTAGAAGAAACAATAGAAGAAAAGCTATGTGCCTTTGGGTTTGGTAATGAATTTTAACAAATGATACAAAAGGTTGACATACACAAAAGAAATGACATTGTGGATTTCTTAATATTTAAAGTTTATACTCTGGAAGACACCTTGTTAAGAGAACAAAAAGACAAGCCACATATTGAAGAAAATATTTGCAAAATACACATCTGAGAAAGAATTTGTCTTCAAAATATATAAAAAAGTATTAAAACTAAACAATAAGTTAAACAGCCCAACTAAAAATGCACACATCTGAACAGACACCTCACCAAAGAAGATCTACAGATGGCAAGTAAAGAAACATCAAAAAGATGCTCAACATACTAGAAAACTGAAAACCACAATGAGATAGCACAGCTGGTCTATATCTCTTAGAACTGCTAAACTCCCTAAAAAATGACAAATTGCTGGAGGAAAAACAAGAACTCTTTTCATTGCCGGTGGAACACAATGTACAAGACCAAAACATGCCACCCCAAAATATAATGGTAGGAAACCAGAATATGCCACCCCAAAATATGTCCCTTTGGCTTAAGAATTATTCCAAGCTGATTATTTTGAAAAAATAAATGCTAACAAAGGAAGTTCTGAAAACAGAGTAGAAGTTACCCTTGTGTAAGGAAAATTTACATCTATAAAGGAAATCCCCATTTAAAAGCTACCTCTCTCTACACCAAGAAGAGAAGGATAACTAAATCACTAAAGAGTCTTTAATAGTGGGATATCTGCCACAACACATTTATACAGATACACAGAATTTTATGGCCAAATGGGTAAATCAAATTCTATTCAAATTAAACAAAATTACTCAGGATGTGGCGTATCCCAGGACAGAATGCATCATGTTGAAAAAGAATTTATGCTACAAATTACTATGGTTTGGATGTGGTTTGTCCCCAAGCCATGTTGAAATTTGACCCCCAATGTGGCAGTGTGGGGCGGTGGGGCCTAGTGGATGGTGTTTGGGTCATGGGGATGGATCCCTCATGAATAGATTAATGTCCTCCATGGGGGTCAGTGGGTACTGCTTCTCATAGGAATGGATTAATTCCTGCAGGAGTAGGTAATTAAAAGAGTCTGGCTTCCTTGGCTTCCCTTTTGCTTTCACTTCTGCTATGTGATCTCTGGTGCACCCCTTGCTCCCCTTCCGCTTTCCACCATGAGGTGAAAAAGACTGAAGCCCCACCAGATGCAACTGCCCAATCTCGGACATTCCAGCTACCAGTATTGTGAGCCAAATGAATCTTTTTCACTTATAAATTACCCAGCCTCAGGTATTCTGTTACAGGAAGCACAAGATGGACTAAGACACAAATGTAGGTAAAAACTCACTGAAGGTGGAGGGAAAATGGTGTTGACCTAAGTCACTTTGAAAATCAATAGAATCTGGAGGCTGAAGGCACATGAACTATACTTCATAATTGGATTACATTTTATAAAGTTATTTCCAACAGAAGCAATTGTGAACAATTGTAAAACCACAGTGTCTGTATCTGGAGTAAAACAATGACTTACATAAGTCGCAGATGGTGGGAACCAGCTTTCTCACTGTTGAAGTGGGAGGTTACAAATTAGCAAGACGAGAAGGCTAGAATGATTCCTGTGATAATAGAGATTGGGAGGATGAAGTCAACGTAAACTTATGCTTAGTTTAATATAGATACACACAGTTCTACATAGAAAACTTTATAATTAGGTGTGTGTAGGTAGGTTAGACACACACATATACTTCCTAGCATTGCTAATGAGGGACAAGATACAATGTGCATTCAGCAGCCAGATGTAAGTTTTCCCACCATTCTGAAAGGAATCAGGCTCTTTGAAGAAATGTCTGATACTAGAACTGGGACAGTATAAATATAGGAGCCAGGATAATCTGGAAGTATCAGAAAGTAAGTACTAAAAAAATTAAAATATATCAAACAAAAATAAAAGCCAATAAAAACAGCTACCGATGGCCAACACAGGAAGGAATTGTGCAACATAATGCTATAGTGTTGAATAATAACTAAAGCTTAAAGTAATTATCTAGGTGTCTGTATTTGTATACCTAGGTGAATAAGCAAATGGAGTTGCATAGAAATCTCCTTTGCAAAAGAATTCCAATAACTGATGTAGACACTCAGGCCATCAAGAAGGTGGAGCCAACTCCTCACTCCGTAAGTGTGGGCTCTGCATAGTGACTTGCTCCAAAAGAACACATGCAGTACGGACAAGGAGGAAAAATAACTTCACAGTGGAGAAATCTGACAAACAGTAGCTCTGCCAAATGATCCAAGTGAACATCAAAGCTGACAGTTCACCTTAGAACATGAAGTGACAATGGGGGACATTCTTTAAAATGCCTGACCAATCCTCCTCAGTGCTATGAAGGTCATCATGAGGTGGAAAGCCTGACACACTGTCACTGCCAGGAAGAGCCTACGTGATGACTACATGTCATGCGGGATCCTGGATGGGATCCTGGGTCAGAGTAAGATAGAACTAAGGGAATCCAAATGAAATATGAACTTTAGTTAATAACAGTCTATCAGTATTGGTTCATTAAGTGTGACAAATTCTGTAAGATAATAATAAGCCATGTGAGACACACTGATTGATAGGAGATGTTAATAACAGAGGAAACTAGGTTGCGGCTACATGGGAAATCTCTGCTTTTTTTTTTTTTTTTTCTGACGATTTCTGTGTAAGTAAAAAGCAGATGTAAAATAAAACTTTATTTAAAACACTTTTTAACACTTCCTTGTTTAATTATTTATACCATGAATTACTAGTAATTGACACTGTTAACTAGTCCTGTTTTTAAAAATAAGAGCATTTATGACACAAAAAATTAAACAGTGCAGACTGATATATAAATCAAAACAAATATTCTTTACACGTTTTCTGTTACAGTAGTAACACATATGTGTAAATTTAAGTATCATATTTTTTTCTTGTGCTATGGTTGTGTCCTAGGTTCATTCTCTAAAATGCTGTTCACCTTAGACCAGGAAAAATATTAACCATACAGACTCTGTTTCCAGTCATAGCTAAATATTTTCAAAAGAGTGACTTTGTAAAAACATGTTCCAATGGCAAATTGATTCATTATGATGGGATCAATTGTACGAGTTCCTGTCTTATTATTTCTTTGCCATGCCTACCTTTTAGCCATAATACAACAGAATCAAATCTGGCCACTGGGAAAAAAAGAAAAAAAAAAGAAAAAAGAAAGGTAGGGTCAGGGAAAAAAAAAGAAAGAATGTGAACAGAACTTATGACCATGATGATTAAATATTTTACCACAATGCTTTCTAAAACAGAAGAGTGTAAAAGGATATTCAAAGTCAATTTCCTCAGCGAGGCTTTGCAGAAAATGAGGAAACTAGAAAAACAAAAATGGCAGGACATTCTATGGGTGATTTTAAATGTTGCTATGTTTTATGGGAAAAAAATACTTTACCTTTTAAAGAATCACAAAGAATTATTGGAAACCCAAACTCTGGAATGTTTCAAATTTAGTTCAGCTTCTATG ''((+8653-8<;?A;5332488<=<=>/-=9:;<:=?A@@<<<<;<<,++++1+;;<=?AD>BAB@11111>@DB?DDEDACECC>AACBD@@??=65555976678;86666778777:98889;/....,***,8;20001;=AF@C@CFA=@ACCD<CDEED:7745;<=B?:;;<@A@;;;78BCEHEICCBFDEBCFSCKA@?A=AAAA>6666<A?8>ACEFHECGEHDDFABD@BEA<===99:?@@ACIBCDCBHDCEA>8:844<>?@BC>>><;@@BCDICB@EEF@BDEEGBDBFGJECFGCSEF@IJECEEE@JIFHJDFA<AD@F@??@=::;;<>10001<>?@BDDLGFFIED@DDCBSICBD=33333455556?ABADFHIA@@A66655;88877;233113-*)()('',.367878?BOSGEGHO8>BDHK@@?A99888?87777=:::99@99888>55545=::999>33323?DDFCEFCDD<9;BEEFEEFEBBACFDEGIGFFB@A@@11111=>B@>>@88555>A:9@@=C<<=:<EEAG@?@>,*6611864=9<>@>B???@;>?>>>?@>>??ACCCBDCB:8778:CDCA@@@AA>>>>>??@?????@??88:111297888977.,,+((/78<AB@@@@AA9++>++BDFHEFDCAA@AB@CDEDFFEDBBCCBAA@?@??EF6320/.--..223?;2223389?@BA?@@@ABBBBB==@??@@?ADDCCD@A?====>ABDFCB33333?9::::D998::<ACMJIFDABBDDCEC@@A?A@9843E>976634112=BDDBBCE?@>??BDIECBABECGEDDBB<<=<;;;99222GIFBA@AAADDCDDABBCCFCAA@@BCEDCA@@@ABA@??@AGEFDKFC<<?EECBCDJSHFHF@?85?FDBFGJHEDFEDEHGFFEFFDFN@?980****)76++++8;<==;764445.---+...=CGGHHMSHKHFBBACCDNEEFEBCDCBDDEFDGGGGEGKED::-)))>FKEEDCEFJGHFFFBCCIHSEFCCDDLDFEFIMEEGG22A@AAADDHEFNIHHEEEESSGD>0000/.-,,-,''(()112226878AGHKGDBD<<GP:8GEFISMJIHIEFDF:54649777789FDFEFEEGFIGSSHLG@=999;@,*,..*'&&'''''((((59>DG99999GA8E98888GFFSKLNIHEGEBB@?7?=@>;;10000244211445??@GGIFSMSSGKLJNKKSJHFHGJKSG@??88999DA?FDGKE;9988:;<7CGHOSHIGIHHEG@89888EGC;730+,<=?FLIIHDFI>=>>CDBAHSSFGFCEELKGLPG222@@???@?;76644;:9.-&&&(%&%%$#$&*))*)+2=BBFGDE==<>GKSMJNSOONDFDFHGCD==6.-++,./211124@BCJMIIMGIMIKGHFFDCCDAA52.--068<778==>@A@DDDDA::;<<SHOS@@?==KLESKJLGHFHLENFSKFSSSKSIEQEAADSCB964-)+'$%%%&9;==FOGJHFHSCGGLGRKKHKLOSSRGIKMGIKNKGKGFMSLF6555656?@?@A@?>@@HJGSSEEIJNOGKIKGGGLNSIFJSKEMGDCCFEIGLF?>::B=<<>=,**)())'''))(((''*--9===AA>?>>>D?>>679980000088=CG@@@?::;<@B64443566;<SJJKPSJKGSGGFHC3KOSHHHNSHLHSSPSSJHGSSSKHSSSSIIJSQSD>A>>>=@@>2A@B@B@AA?AJHIKRSHHHGJJSJSSSNISNFHSJSLSSSKJ?CDIDHHSKPGSI>?>>ASSHIIKSSFFFFJJIJQISSSSFKFGMDCECCLFCCEEISQSMSJISBG89IGOKONSSSIJPHJNHQJLJSF872//--20-'((::<:DCHESHILDHSLD::::AHGSIOJJLISSLHJMHJIQGJHJKSOIGHGKKHHHSISSMJLHIED>@EHACBANM@<=BA8=))((()22201255?CJGHHJHSLJLSNGHSDDCBB42222ESSKSKSKGQISGKSSHISSEKJSKJRSQJSISKIIOHISSSLISJGSMQJEJBAA???===::CCBADCEDDBCBA777778AANGKKHFGDDGISJJSKPHIKLGKJSSKFGSMSSHKOOKIJGDB655555>;;;;GIHSSOQKJISGQIELHGEDHHRJGKLKKNISJSOKSLLSPFSMSJSPSSISF<-,*(()%$$#$')$$$$&&&'',4<>AEKFIEF>GCGB==>?9><=C977788DA:8SSMNIHFJELEEEFGEFMMSISJIFJHKGSJFFQLSISKSLFGSH===<<<B@CSJNLELGGHLDD@?AFHFHGESSGGJFIEEGGSLPKNLQMKSJKMRFSISHQHNKLSSKSPSSHGJIIE;63-7FHONHE2111-DGFSEHKIHSDCFCECORSSSMSSSLJISIISHIGKKSKHIGFDCE7.++,/1899:>DFFDDFSHIJF?@A@BIKJSSLJLMGSMSSJSSSJED889SSSPSSSSLJMRHHFGGSJHSKNSSSGSLISBABAAMSESG>====GECBEBGHBMSNMMAABIEOMGSKOSSLSJSSKKLNHJMLDE0000/1/..//45444459:888777=GEHMGNIGLIEFJKJSSLSSKSNKNSRFSIFJA@AAPCNJDA>===>AESIIEEFJISJEEGSSSJKSFSC>D?CDEHFC????>:--555>?@EB9988<<B>>=4.-,(**489:CEKSGFRSJGJSSKGS@@@FBAFEHSHISSHFB@@ISJSLLOSSLSSNSKSHSSCCGISIJSLSSDDDEMSLSJHHIISNSHSISMKOSMSSSNSKSKSHSSSKSOLSLPOILHSIKLJLGKQPIIJJHKMSIKMSSMGLJPSSGSLHEILIHGSISHNISISFDCEA>('''::KFEGMLSNNNSOJGLEOJEGIGBA(&&&'):DJFSFIFPHEFIGGIISJHMGLSKFHFJPOFOJSSNSSBIEHSHISKSIJLRCECFISFSSHPJSFIS=888==AB?;;<<GFEKLKMIC:4333=>ABBCGSL=:<FSLHSKIESLJKNGH9975:<<<>BC@CHFA<==<???EJFJIFKLKPIIQJKSMJJHSFDFEG888888IFFHKEE=====LIKFSMHHGEGCECHHGHKIMSLSHSHNKSPSMSKJSHSKISSJJISHLGSGIIGIGNSHIMCCDGSSMSHJILSKHNOJPNNSHDCDLSQKSKGCEDEJSNGFFIDECEGFGIHSJSSB??>425;8:?ACECEHBBA7('''';;;;<EJHHLSKSSSHSJSKSIHIHGMED====?ILE?ABB>GEGDIHGH::652CCEDDDFDDFGJIIFLJSLPSMGOH;:,++(&&&(./:;;;;=BQSKHKSSLLMQNKKSJKSHGFESFSGISOHSSLSJIHEHJKORSISJHQILSIGJSSSSSJKLSSSLIH=<<<<=ALEEC<:89:DFSSSJSHMSNJHSKGGSSQFGDFJ<=<<<87889FJJGGPSKSRSEJNISSHIOSLDDHGKS;;;:;?A@BFE>2=@AJGGGELHGKLSOKHSJFJNSINJHGJJGC:99:<GLF9(((((?E;9666@>??SHSKSKNJHDCCEEGGOGMMSISLISHSJSLHSGSJMSJLNJSHOSNSMDDBAEJ><<99;@ACBFDFDISJJOHHMSQSHKHSNSSHKMBAA><===CCFKEREFCCC4444(('&%%%$$$%&&'/21)()()*8'<?8''DIFGSIIGHSNLSH@SSHNIRHLEJGMED31111::98''''(JSGICDDCKGIF=982/.,+33=EGDCBDDAD6/.-+,+)())+,22228?C>D=<<=;AEDMIKSGSLHFSHSPJMGJFIGESSEG?<;8898*)))..../58:9:EMHSHGMILD@??@AC///01////(((-*HGSFJSLMSIHSSGGFGIEKMCDACDCCCDBGFFCGDGFFFEFEGEKIJHECCBCGJFEDEFGSHSJHGFEFHGMGDHGFFGGMGLHHMSNSGHHHPFSFFIHJFSNKIGI@1AACDB?:++++,./<>?D?9KIDFEKECCCED?B=94-++,..<;;66666<;22226:=>?@B((((2;<++++++>==ABB??B RG:Z:A  MM:Z:C+h?,174,42,186,70,21,45,18,53,9,4,49,36,10,47,3,22,6,27,35,35;C+m?,174,42,186,70,21,45,18,53,9,4,49,36,10,47,3,22,6,27,35,35; ML:B:C,1,212,4,16,25,3,1,33,1,19,4,3,3,3,19,10,16,2,18,1,254,42,1,239,229,252,0,222,254,1,1,252,1,1,236,244,2,1,237,254 NM:i:49 ms:i:647    AS:i:618    nn:i:0  de:f:0.0649652  tp:A:P  cm:i:20 s1:i:137    s2:i:119    MD:Z:15^C32^T20^C11^C25C8^C29^C33A29A5A17^T5T5T2A3^TA0A0A3^T5T57^CCTAACCCCTAA3^T13^CCCT29T2^C52 rl:i:396

Sample_5663A-1-4243P.guppySup.meth_haplotype.log

[vahidAK]

Hi @weishwu , Can you run it with -of methylcall,bam2bis, and another time with -of methylcall to see if it works? As you mentioned, there might be tags in bam causing this issue.

[weishwu]

@vahidAK I reran it with -of methylcall and it ran through without issues.