rf-count-genome outputs 0 % mutated alignments - Githubissues

dincarnato / RNAFramework

RNA structure probing and post-transcriptional modifications mapping high-throughput data analysis

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rf-count-genome outputs 0 % mutated alignments #49

Closed mohamedbahru closed 8 months ago

mohamedbahru commented 8 months ago

I have the Oxford nanopore sequencing data.

I have done the alignment with splicing using Minimap2 aligner.

Then I used rf-count-genome function to calculate the mutations per base.

I got the 0 % mutated alignments. Why?

The output was displayed as follows.

[+] Making output directory... [+] Guessing file types:

[4m[1mSample[0m [4m[1mType[0m [4m[1m5'-end trimming[0m [4m[1mLibrary type[0m

sorted BAM Ignored unstranded

[+] Importing chromosomes from reference, and building count table base structure... [+] Inspecting SAM/BAM file headers... [+] Sorting SAM/BAM files...

[-] Sorting sample "sorted" (PID: 62495)

[+] Calculating per-base mutation counts and coverage. This may take a while...

[-] Processing sample "sorted" (PID: 62622)

[+] Statistics:

[*] Sample "sorted": 54 chromosomes covered - 0/7671926 (0.00%) mutated alignments

[+] Cleaning up temporary files... [+] All done.

Could you please help me? Thanks.

dincarnato commented 8 months ago

Can you please paste below 2-3 representative lines from your BAM file?

mohamedbahru commented 8 months ago

[mohamedb@binf cDNA_no_UV_crosslink]$ samtools view sorted.bam | head -5 7183e3b4-9a78-440c-b1c2-4629c10c24d3 16 chr1 3053489 60 1444S48M2D2M1D17M2D17M1D17M1D6M1I4M1I4M1I19M4D10M2D3M1D10M1D6M5D9M1D1M1D19M1D25M1I5M1I2M1I4M2I2M1I6M1D6M1D13M1D24M3D3M1D4M2I8M1D3M1I43M1D2M1D12M1D11M1D8M1I9M1I7M2I2M1D4M3D12M1D9M1D6M3I9M3I5M4D2M1D28M2D2M1D5M1I12M1I8M5D10M1I11M1I9M1I14M2I33M1I13M1D17M1D4M1D4M3D18M1I4M2I4M1I22M1D7M1D7M1I4M4D7M1D4M1D7M1D5M2D19M1D27M1D2M1D1M1D5M3D24M1D6M1I10M2D4M3I23M1D15M1I13M3D22M1I64M2D10M1I2M1D7M1D14M1D5M3D21M1I4M1I4M1D19M1D25M1D6M2D10M1I10M1D10M1D19M2D2M1D2M2D8M1I12M1I4M1D11M2I4M2D10M2D2M2D18M1D10M1D20M1D11M1D13M13D47M3D15M1I56M1D7M1D4M2D30M1I6M2D22M1I11M34S 0 0ACCAGCATTGGCTATCTCCATGTCAGGGGGATAGGACAGTAAGGCAAGAAAACACAATGAGGAGATATAGCTACACTGCAGCTTGGAAAGCGGAAATACACCCATCATCACAGAGCTTCCATAAGATGGTACATATACATATGGACAGATCAACCAAGTCACCTCATGCCAGGCACGTGTGTGGGTGTGTGTGTGTGTGTGTGTGTGTGTGTATTTAATGGTAATTTGAAAATCACATACACATATCGATAAAAATCGCATGGAAGACACTCATCATCACTCATAAGATTGGGAGAAACTGGAAACCATATTCTGCTGACTGGATTGGATGGGTATTTTCAAGGAAACATAGACATGTGGAAAGAGGAAAGAAATAGATGCTCATATTAGTGCTGTGAAAAAGAAATGGTACAACCACTGCAGAGACACAGTGTGGAAATTCTCAAAGATCTAGAAAACTGTTGCATAGACCCAGCCATTTCTTTACTAGCCATATATAGACATTGTTGGGTACACCTTGTTAATAATATATTGGGTTATAAGGAGCTAAAGACTACCATATCCTATGTATTCATGCATAGATAAAATTCTATAAATATAATAAATGATATAGATATGTGGAAAGATCATCATTCTCAAAAGACTGGATGTTGGTACTTAGATATAAATGGTTATGGTGACAAAACCACATAAAACTGAATACAATTTCATAAACAAATATATATAATTGGATCTTATACAAAGAAAATAATAACATAGATAGAAAATAAGAGCATAGAATAGGACACAAGAGTAGTATGGAAGAACAGAAGAGATATCTGTTGTTTCATAACCTACGGGTTCCATTTGAGGATGATAGGATGTTTGAGGAAGATAGAGACGGGCTGACAATACATCCCACACTTAAAACTGATCTACTATACACTGGGTGGTTGAGAAAATGCACAATCTTTCCTTGAAAAAGCTATAGCTGATTCCATTAGTGACCATCACAGAAACTGATTCTATAACAGTCCATCAAGAAAGACAAAAGATCTGTGGCACAAGTTCAGAAAGCAAGTCTCAGCCAAGGCCTAGTTGCAACATCTACACAATGGATGGTCACTGTAAGGAGACATCAGGCCACTGTGGCAGAGACAAGAGGCTGAATCTGCCTGGAGACCATACAGTAACAACAAGATGGGAACAAAGAGAGGAGATACAGGCTCCATTTTTCATTCCAATGCTGTTATTATCTCAAGGCAGGAACCATGAAAGCTCAGGAAAAGGAAGGGAATCTAGGAACACTGTCTCCACATAAGACATCTTACTGTTATTAAAACAGGGAAGGTAGAGGGCAAGGATACATAGTCAACAAATGACCTGGAGTTCTATAAATGGCTAGGACATGCCTTCCATGGCACGTGATCTGTCTTTACACTGTCCACTGTCACAGTTGGTTGGCAGCAGAGTGACAACCTAGACAGGGGACTGTGACATCAAAAGAGTGTTACAAAAACATGGTATCACCTAAAGCATGTCCTAGCCATTTCTGGAAAATGGAAGCAAGTTCCTTTCCACTCATGCATCCTTCCATCTCCTCTGCCTCCCAGACAGAATGCTGCCATGTGAACATGTTGTGTATCCCAATGTGTTTCCTGACCTACATGATACTGCCTTGAGCCTCACTACTGCAACATGCGCTGCATGAAAGTGAACCTGTATCCTATCTTTGTTCCTATCTCTGTTGTTATCTCTGGGTTTTCTAGGAGGAACTGAAGCCTTTCCCTGTCTCTGCCACATGATCACCTGATGTCCCACAAGAACCATGGACTGAATTGTTTAGTGTTGGAAACTGCCTCTTGGGAATCTAGGGCTCCTCTTTGCTAACTTTTATTGGAGAACTTCCTTGTCTGACCTACTACGTTAATTATACCAGAGTTCACTGTGATGAGTGAATGGGAATAGCTCATCTGCTTTTCAATAAAGTTAGCACCATTCTGATCCTACCCAGCTAGATAGTCAGTGGTAGTTAAGTGCTTATAATGTTGTCATTATCATTTCTTATCTATTTCTAGACATTCTCATCATCCTCAATGAATTTGTCTATGAAAGAACACACTCTGTCTCTTCCTGTTCTTCCATACTCTTGATATCTATTCATATTCTGATTTGTCTACATCCATTTCTATAATAAATCCAATCTGTGTACCTTCTAAATTATGGAAAATCTATACATTCTACTATTGTTCATAGCATAATATTGTAAGGTTCATCCTTTCTGAAAAATACGAATAACTTCATAGATCTTAAGAGATGATCAGATTTGACATATCTAACACCATTTATTATGATCAATTTATCTATACATGGTATGCATGGGCTGTGGTCATCTTTAAGTGACCATAATGAATGCTGTTATTAACAAGGTGTACGTATATGTTGAGGCTCTTGCTTTGATTATTCTGTGCATTGGCTAGAGTTATTGCCTAGTCACACTGGTAAACTTGAGTGTTTTAAAGTACCATGTGTCTTCTGCAGTGGCTATATCATTATTTAGCAGCACTGAAATCTGGCATTGATTTCTTTCTCTTTCACATGTATTATGTCTGAATTGCTATTCGAGAGAGATAGAAGACAGAGTCTTGTGGAGTTAGTTATACTTCCTACTGAGTGATCATGAGTACTTTTTATGAATTTCTGGACATCTGTATGAATTTTTGGAGAATTTCTATTTAAACACACACACACACACACACACACACACAGATCTATAGATTGAGGGAGGGATTAGGTGTTTGTGTTTATGTATTGATATATGTATATGTACATATTTATGAATCTTTGATAATAATGGGTTCTGTGAGGGTTCCAGAGCTTTCAGTGTTAGCTATCTCTCTCCTACACTGTCTTTAATCTTGCTGTCCTAATCCCTGACATTCATACAGATGCAACTGAACGAAGTAATAGGCAT 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CCATGGAAGGCATGTCCTAGCCATTTATAGAACTCCAGGTCATTTGTTGACTATGTATCCTTGCCCTCTACCTTCCCTGTTTTAATAACAGTAAGATGTCTTATGTGGAGACAGTGTTCCTAGATTCCCTTCCTTTTCCTGAGCTTTCATGGTTCCTGCCTTGAGATAATAACAGCATTGGAATGAAAAATGGAGCCTGTATCTCCTCTCTTTGTTCCCATCTTGTTGTTACTGTATGGTCTCCAGGCAGATTCAGCCTCTTGTCTCTGCCACAGTGGCCTGATGTCTCCTTACAGTGACCATCCATTGTGTAGATGTTGCAACTAGGCCTTGGCTGAGACTTGCTTTCTGAACTTGTGCCACAGATCTTTTGTCTTTCTTGATGGACTGTTATAGAATCAGTTTCTGTGATGGTCACTAATGGAATCAGCTATAGCTTTTTCAAGGAAAGATTGTGCATTTTCTCAACCACCCAGTGTATAGTAGATCAGTTTTAAGTGTGGGATGTATTGTCAGCCCGTCTCTATCTTCCTCAAACATCCTATCATCCTCAAATGGAACCCGTAGGTTATGAAACAACAGATATCTCTTCTGTTCTTCCATACTACTCTTGTGTCCTATTCTATGCTCTTATTTTCTATCTATGTTATTATTTTCTTTGTATAAGATCCAATTATATATATTTGTTTATGAAATTGTATTCAGTTTTATGTGGTTTTGTCACCATAACCATTTATATCTAAGTACCAACATCCAGTCTTTTGAGAATGATGATCTTTCCACATATCTATATCATTTATTATATTTATAGAATTTTATCTATGCATGAATACATAGGATATGGTAGTCTTTAGCTCCTTATAACCCAATATATTATTAACAAGGTGTACCCAACAATGTCTATATATGGCTAGTAAAGAAATGGCTGGGTCTATGCAACAGTTTTCTAGATCTTTGAGAATTTCCACACTGTGTCTCTGCAGTGGTTGTACCATTTCTTTTTCACAGCACTAATATGAGCATCTATTTCTTTCCTCTTTCCACATGTCTATGTTTCCTTGAAAATACCCATCCAATCCAGTCAGCAGAATATGGTTTCCAGTTTCTCCCAATCTTATGAGTGATGATGAGTGTCTTCCATGCGATTTTTATCGATATGTGTATGTGATTTTCAAATTACCATTAAATACACACACACACACACACACACACACACCCACACACGTGCCTGGCATGAGGTGACTTGGTTGATCTGTCCATATGTATATGTACCATCTTATGGAAGCTCTGTGATGATGGGTGTATTTCCGCTTTCCAAGCTGCAGTGTAGCTATATCTCCTCATTGTGTTTTCTTGCCTTACTGTCCTATCCCCCTGACAT ''+,-..55776610//..//(((++'+&%%$$$$$$%&'(''''%%&%%%$$$$&''&''(&&&&&%&&'''(()+(),,)(%$$'&&&%%%%$$$%()))(+,-,,+)&%&'(()*(((''('()()%%%%%()++('%%%&%$$%%&)''&&'''('&%$$$$%%%$$%%&&))(&&&&')*()'&',)+++'((+)&&&&&+()')')))&&''(,,,-0.('$$%%%&&&&))('%$$$#$$%)*..,,,-./,(&$$%&(+-/144420.)(''$%'('$$%%%%%%%$%%%%&$%%%$$&&&&(((((((()+(&&$$$$&&%&(,(+&%%$$$$$%'((&%$%%&(+-+'&'((''&%$$$(,('%$%&'')+)++-,,.//+))$$$$#&)*.-,+((((('&&&&(*,,.'''(-,'&%%%&&'')(('&&'&&'''',+(,,,,,/'&&%&&$$$%&&&&%&('('&%%$$$%&%%$$$####$$%%&(*,,&'&&'''*1--.)'&&'&%%%&&&(-('%$$##'(()))((&&''()+++,,,,,+)&&&'(+,,(())--.*(''&'&$$$$%%%&')(('('&&&&'()-0,%%%%$%%%%&&%%&&&')0,--+&&&&'-*)***((&&&&$$##$%%%&&'0+&&&'&%%%&%&&&&(((&'&%%$$$$$&'$$$$%(&&&((()'+%%%&%$##$$%&&()&&&%%%%&'%$%$$%$$%(&%%%&),+--+)(,+'(((()*,,(((''(())())((('((''%%$$%)&''''&$$$%%%%%'''''&'%%&'$$$$$$%%$$%''('')((%%%%&'%%''&%%$''''&%%%%%&&''(%%%%$$$$%%$$%$$%%&'(()-..('&%%$%%)$$$$%$%&'&*))))(''(%&&%%%%%$&%%)%%(((&%'%&')'&$$######$$%'%%&&()''&''+,,(&&&'&&%$$%%&&)++0/.,,,./---.8720.+++)&'('(('&&''()'&&%$%%'&''%%%%$$$%&&'()(%&&'+,-,++)())'(&&&&'(''&&&&&'()./114,+%%&&&'(&%%&('('(()))))(''''(''&'%$$&'+('''&'''&'(&&'()/469::;;EHKSKIIIKHFSIHHQDCB54444><<41/.,+((''/0/..''''&%&(+(((((&%$$$%&'))%%&''('(&&%%&&$%&'&))))&%$$&%&&''''&&&&&('&%%$$$%&%''%%$###%&%%$$$$$%&''&'&&'&&&%$%&&%%##$%%%&%'$$$%%'((+1/0/00122223 NM:i:419 ms:i:367 AS:i:293 nn:i:0 ts:A:+ tp:A:P cm:i:10 s1:i:110 s2:i:0 de:f:0.2076 SA:Z:chr1,3053489,-,1444S1475M86D34S,60,353; rl:i:99 1f6d3106-86b3-410a-9e47-236ad099f307 2048 chr1 3055661 37 48H6M598I167587N3M1D1M1D10M1D6M1D7M2D26M1I12M1D5M1D13M5D11M2D2M2D8M1D3M2D5M2I9M1I19M2D10M2D9M1D21M1D7M1I3M1D8M1D15M1D10M1D62M3I6M1D12M1D9M2I9M2D16M4I4M1D3M1I3M1I17M1I23M4D33M2D5M1D32M1I10M1I2M1I14M1D9M2I3M3D1M1D22M31H 0 0 CTATCTTTCTTTTTTTTTTTTGCCTTTTAAGAGTAAACTAAGATTTTACGATACCTAAACTGATTGATGACTTTTCATTGACAAAACATAAATACTGCATTCTGAAGGGGCAGTCACTAATTGCACTTTTGATCAAATTTGTAAGAACAGTCTGACTGAAGACTACAAAGATTATTAGAGAGTGGCATGTGCATGATTTATTAATTTTCAAGAAATACATCAGCATTGTAATTTAGAATTGATAGTCTGTGAGAGATGAACAATTAAGCAATTTACAAGTTCGGAGTATGACGTGGTCTGATACAGATTAAGGTTTTGATTCTTGAGCATTTTTAAAAACCTTCCCCATCTACAGCACACAGACATACATCTGTGCTCAAACCAGGGAAGACTGTCTCTAACTTCACCCACAGGAAGCTAGTTTTGGGCCCAGCATTGGGCAACAGATGGCTGGGAGGAATGAGGAGAATGACTTTTCAGATGATGTTTTTTTTTTTGGCAAAAAATGTTTTCGGCACAACAATTTTAATTACAATTGGATGACAATTATGAGAGCTTTGTTTTCAGAGACTTTGTGGTAAAGCAAAAAACTCTCCAAAAAAATAAACTTTATAATTGTATTAATCATAATAAAATCGGTTGTGCCCAAAACAATTCTTTTCGCGAAAAAAAAACGTGTTGTAAAGTCGCTCATCCTCCGCATGGTTGGCCATGCTCCACCAACTAGCTTTCTGCTGAGCAGAGTCTTGACACAGTTTCCTGTTCCACACAAATAATCTGATTTTGTGTGTGGATGGTGTTTGAAATATGAACAATCAAACCTGAGTTGTATTTGATGCTCAATGCCATATCTATGAGCTCGCAATTGCTAAAAATATTCATCCCTCAGATACTGTAATTATCAATTTACAATTGTTGTGCATATTTTTCTGGTTCAAGTAAATAGATTTTTGTGGCACTGTCGGCTTCTTAATAACTTTGCAACCCCTTCTAGTTACAACTGCTCCTAAAAATGAATTAAAATGTGATTAGTCACTTCCCTTACACCAAGTGTTTGAAATGTTTTGTCAACTAGTTATTTAATTCAAAGAGTTTGGTATTGTAAAATTTAAAACTAGTTATTTAAAAGGCAAAAAAAAAAGAA ,,(')'&%%((),03446<=21/../-)(''))%%%&%%$%%%%0,.(''%%''&(('(&&%)((((((((((''('(---.0///)))(((&%%%%$#$&''(((&&&$$$$$&&'())))+,+&&'&%&&$%&#####%%$$&+&&%%&&%&(')&&%%&&'()'&&%%&&&&)((()).))((&''&'()&$%%%&%$%&%%&&&(((('('&&&))&%%&-'&&&'().1('''',+++/.12((''&))))))()))'&''(&&(%%&&(('''+''%%&$%%%%%%(&&''(&%%%((+,&&%''$$&'())+))),,+++&$$$'(-+++++.-.1/--(''&&'(()''(('-,(&%&%&+)&'(&%&&)('&&&&%&&&&(-(+32+''(+,(''(()(),,01.+(((&&()((('&&%&'145545<50(&((''&%('&&&''/)(''&()).//3456;CE1100333756,,,++'%%%%)),,-11,2/.++'&%$$$$%%''''&%$$$%&%%&''')&'''/%$$%&))())/(''%%(&&'')+,,&%%$#$$%&)+,,)('%&&'&%%%$$$$%&)),('&%$$%)))&&'''(&''&%&&'&&())124'&&%&()/-(&&%&%(%$$%%)++,++)))+'%%%%&()&').3-(&''''%'&&,.--,(/,)('&%$%%&'&&'(%&&&),,,+)()+,)))+))()(&&&&%%%$$%'&&'((&&%'&&&%$$$%&%&''&%$$$$'((+&&%&+.)''&&%$%&&+%&0....-.//06.-,,)(&&&(()&&%%&'&&'(()))+(('&%'$%%')/.-+'&&&(&&'(''%%%%&,((-,,,,**)+*,,,-2)'&%&&%&%$$$%%&&'&&&)((%%%&'(&&%%%%%$%&&'&&&&+'&&&('&&%$$%%&'&&&'(,*++,0,,+((('(,/47(''')))0/.'%$$%'))))))&''''',(((&''&+))((,.-.)%%%$&%%%%%&,+'),,)))%(%%&-''((001110112-&')(''%$$$%&&(-,+01///.,,-,(&&& NM:i:725 ms:i:201 AS:i:125 nn:i:0 ts:A:+ tp:A:P cm:i:13 s1:i:95 s2:i:0 de:f:0.181 SA:Z:chr5,89354138,-,31S547M28D645S,1,114; rl:i:45

dincarnato commented 8 months ago

Your reads miss the MD tag. Please run "samtools calmd" to add it, and rerun rf-count on the resulting BAM file.

Danny