Assertion failed: (readCnt > 30), function GetAlignmentsInfo, file ./blocks.hpp, line 554.

[CristianRiccio]

Hi,

I used rascaf like this:

rascaf -b Aligned.sortedByCoord.out.bam -f draft.fa but I got the following error:

Found 135172 exon blocks.
Assertion failed: (readCnt > 30), function GetAlignmentsInfo, file ./blocks.hpp, line 554.
[1]    76830 abort      rascaf -b  -f

Can you help me solve this problem, please?

Cheers.

[mourisl]

Could you please show me the first few (say 20) alignments from the Aligned.sortedByCoord.out.bam file?

[CristianRiccio]

L180:314:H57G7ADXX:2:1205:16224:67007 256 tig00000002_pilon 9850 3 100M 0 0 CCTCCTTTGGTGCTCCATCTTTGGCAGCCTGCTCCCTTCATTTCACGTCCTCCGCCAGCTCCATCCAACCACACTGGTCCCTGCTGATCCCTGCTGCTCC BBBFFFFFFFBFFFFIFFFFFIFFFFFFFFFIIIIIIIIIFFIFFFFBFBBFFIIIIFFFFF<BFFFFFFFFFFFFFFFFFBBBBFFFFFFBBBBBFFFB NH:i:2 HI:i:2 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:1205:16224:67007 256 tig00000002_pilon 9850 3 100M 0 0 CCTCCTTTGGTGCTCCATCTTTGGCAGCCTGCTCCCTTCATTTCACGTCCTCCGCCAGCTCCATCCAACCACACTGGTCCCTGCTGATCCCTGCTGCTCC BBBFFFFFFFBFFFFIFFFFFIFFFFFFFFFIIIIIIIIIFFIFFFFBFBBFFIIIIFFFFF<BFFFFFFFFFFFFFFFFFBBBBFFFFFFBBBBBFFFB NH:i:2 HI:i:2 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:1205:16224:67007 256 tig00000002_pilon 9850 3 100M 0 0 CCTCCTTTGGTGCTCCATCTTTGGCAGCCTGCTCCCTTCATTTCACGTCCTCCGCCAGCTCCATCCAACCACACTGGTCCCTGCTGATCCCTGCTGCTCC BBBFFFFFFFBFFFFIFFFFFIFFFFFFFFFIIIIIIIIIFFIFFFFBFBBFFIIIIFFFFF<BFFFFFFFFFFFFFFFFFBBBBFFFFFFBBBBBFFFB NH:i:2 HI:i:2 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:1205:16224:67007 256 tig00000002_pilon 9850 3 100M 0 0 CCTCCTTTGGTGCTCCATCTTTGGCAGCCTGCTCCCTTCATTTCACGTCCTCCGCCAGCTCCATCCAACCACACTGGTCCCTGCTGATCCCTGCTGCTCC BBBFFFFFFFBFFFFIFFFFFIFFFFFFFFFIIIIIIIIIFFIFFFFBFBBFFIIIIFFFFF<BFFFFFFFFFFFFFFFFFBBBBFFFFFFBBBBBFFFB NH:i:2 HI:i:2 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:1205:16224:67007 256 tig00000002_pilon 9850 3 100M 0 0 CCTCCTTTGGTGCTCCATCTTTGGCAGCCTGCTCCCTTCATTTCACGTCCTCCGCCAGCTCCATCCAACCACACTGGTCCCTGCTGATCCCTGCTGCTCC BBBFFFFFFFBFFFFIFFFFFIFFFFFFFFFIIIIIIIIIFFIFFFFBFBBFFIIIIFFFFF<BFFFFFFFFFFFFFFFFFBBBBFFFFFFBBBBBFFFB NH:i:2 HI:i:2 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:1:1102:12210:37984 16 tig00000002_pilon 11564 3 100M 0 0 CGGGCCTGCGGCCCTCAACCTGATTTATTGGGCTTAGAGTAGGTTCAACGATTGTCCAACACTTCTATGTGTATTCGGGCCTGCGGCCCTCAATCCGGAT BFFFFFFFFFFFBFFFBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFIFIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:2 HI:i:1 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:1:1102:12210:37984 16 tig00000002_pilon 11564 3 100M 0 0 CGGGCCTGCGGCCCTCAACCTGATTTATTGGGCTTAGAGTAGGTTCAACGATTGTCCAACACTTCTATGTGTATTCGGGCCTGCGGCCCTCAATCCGGAT BFFFFFFFFFFFBFFFBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFIFIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:2 HI:i:1 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:1:1102:12210:37984 16 tig00000002_pilon 11564 3 100M 0 0 CGGGCCTGCGGCCCTCAACCTGATTTATTGGGCTTAGAGTAGGTTCAACGATTGTCCAACACTTCTATGTGTATTCGGGCCTGCGGCCCTCAATCCGGAT BFFFFFFFFFFFBFFFBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFIFIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:2 HI:i:1 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:1:1102:12210:37984 16 tig00000002_pilon 11564 3 100M 0 0 CGGGCCTGCGGCCCTCAACCTGATTTATTGGGCTTAGAGTAGGTTCAACGATTGTCCAACACTTCTATGTGTATTCGGGCCTGCGGCCCTCAATCCGGAT BFFFFFFFFFFFBFFFBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFIFIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:2 HI:i:1 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:1:1102:12210:37984 16 tig00000002_pilon 11564 3 100M 0 0 CGGGCCTGCGGCCCTCAACCTGATTTATTGGGCTTAGAGTAGGTTCAACGATTGTCCAACACTTCTATGTGTATTCGGGCCTGCGGCCCTCAATCCGGAT BFFFFFFFFFFFBFFFBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFIFIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:2 HI:i:1 AS:i:98 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:1:2109:5173:7744 256 tig00000002_pilon 12291 3 98M 0 0 GTTCCGGGCATTGAACTGGAGCAGGAGCAGATTCCGGAGCAGGTGCAGGGGTCGGTTGTTCGAGTTGTTGAGGTCGAGCAGCTGCAGGCTGTTCAGTA BBBFFFFFFFFFFIIIIIIIIIIIIFIIIIIIIIIIIIIIIIIFIIIIIIIBFBFBFFFFFFBBBBFBBFFFFB07<BBFFFFFFFFFBFFFFFFFFB NH:i:2 HI:i:2 AS:i:94 nM:i:1 RG:Z:RG1 L180:314:H57G7ADXX:1:2109:5173:7744 256 tig00000002_pilon 12291 3 98M 0 0 GTTCCGGGCATTGAACTGGAGCAGGAGCAGATTCCGGAGCAGGTGCAGGGGTCGGTTGTTCGAGTTGTTGAGGTCGAGCAGCTGCAGGCTGTTCAGTA BBBFFFFFFFFFFIIIIIIIIIIIIFIIIIIIIIIIIIIIIIIFIIIIIIIBFBFBFFFFFFBBBBFBBFFFFB07<BBFFFFFFFFFBFFFFFFFFB NH:i:2 HI:i:2 AS:i:94 nM:i:1 RG:Z:RG1 L180:314:H57G7ADXX:1:2109:5173:7744 256 tig00000002_pilon 12291 3 98M 0 0 GTTCCGGGCATTGAACTGGAGCAGGAGCAGATTCCGGAGCAGGTGCAGGGGTCGGTTGTTCGAGTTGTTGAGGTCGAGCAGCTGCAGGCTGTTCAGTA BBBFFFFFFFFFFIIIIIIIIIIIIFIIIIIIIIIIIIIIIIIFIIIIIIIBFBFBFFFFFFBBBBFBBFFFFB07<BBFFFFFFFFFBFFFFFFFFB NH:i:2 HI:i:2 AS:i:94 nM:i:1 RG:Z:RG1 L180:314:H57G7ADXX:1:2109:5173:7744 256 tig00000002_pilon 12291 3 98M 0 0 GTTCCGGGCATTGAACTGGAGCAGGAGCAGATTCCGGAGCAGGTGCAGGGGTCGGTTGTTCGAGTTGTTGAGGTCGAGCAGCTGCAGGCTGTTCAGTA BBBFFFFFFFFFFIIIIIIIIIIIIFIIIIIIIIIIIIIIIIIFIIIIIIIBFBFBFFFFFFBBBBFBBFFFFB07<BBFFFFFFFFFBFFFFFFFFB NH:i:2 HI:i:2 AS:i:94 nM:i:1 RG:Z:RG1 L180:314:H57G7ADXX:1:2109:5173:7744 256 tig00000002_pilon 12291 3 98M 0 0 GTTCCGGGCATTGAACTGGAGCAGGAGCAGATTCCGGAGCAGGTGCAGGGGTCGGTTGTTCGAGTTGTTGAGGTCGAGCAGCTGCAGGCTGTTCAGTA BBBFFFFFFFFFFIIIIIIIIIIIIFIIIIIIIIIIIIIIIIIFIIIIIIIBFBFBFFFFFFBBBBFBBFFFFB07<BBFFFFFFFFFBFFFFFFFFB NH:i:2 HI:i:2 AS:i:94 nM:i:1 RG:Z:RG1 L180:314:H57G7ADXX:2:2202:3169:9608 16 tig00000002_pilon 16659 255 30M163N71M 0 0 ATTTATGGATCCATGGCAATCGGCGACGAGCTTCGCAATGGAAGATGAAGATATGGGCATGCCATCTGGAAAATATGCTCGAATGGAAGATGAAATGGGAG FFFFFFFFFFFFFFFFFFFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:1 HI:i:1 AS:i:99 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:2202:3169:9608 16 tig00000002_pilon 16659 255 30M163N71M 0 0 ATTTATGGATCCATGGCAATCGGCGACGAGCTTCGCAATGGAAGATGAAGATATGGGCATGCCATCTGGAAAATATGCTCGAATGGAAGATGAAATGGGAG FFFFFFFFFFFFFFFFFFFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:1 HI:i:1 AS:i:99 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:2202:3169:9608 16 tig00000002_pilon 16659 255 30M163N71M 0 0 ATTTATGGATCCATGGCAATCGGCGACGAGCTTCGCAATGGAAGATGAAGATATGGGCATGCCATCTGGAAAATATGCTCGAATGGAAGATGAAATGGGAG FFFFFFFFFFFFFFFFFFFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:1 HI:i:1 AS:i:99 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:2202:3169:9608 16 tig00000002_pilon 16659 255 30M163N71M 0 0 ATTTATGGATCCATGGCAATCGGCGACGAGCTTCGCAATGGAAGATGAAGATATGGGCATGCCATCTGGAAAATATGCTCGAATGGAAGATGAAATGGGAG FFFFFFFFFFFFFFFFFFFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:1 HI:i:1 AS:i:99 nM:i:0 RG:Z:RG1 L180:314:H57G7ADXX:2:2202:3169:9608 16 tig00000002_pilon 16659 255 30M163N71M 0 0 ATTTATGGATCCATGGCAATCGGCGACGAGCTTCGCAATGGAAGATGAAGATATGGGCATGCCATCTGGAAAATATGCTCGAATGGAAGATGAAATGGGAG FFFFFFFFFFFFFFFFFFFFFFFFFFFFIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFFFFFFFFBBB NH:i:1 HI:i:1 AS:i:99 nM:i:0 RG:Z:RG1

[mourisl]

It seems this data is single-ended. Rascaf is designed for paired-end data set.

The assertion happens when it tries to decide the insert size of the mate pairs and could not compute the size because there is no mate pairs.

[CristianRiccio]

Thanks. I did not know Rascaf was for paired-end data only.

[emmannaemeka]

hello, i also seem to have similar problem Found 432405 exon blocks. rascaf: blocks.hpp:556: void Blocks::GetAlignmentsInfo(Alignments&): Assertion `readCnt > 30' failed. /job_scripts/3643772: line 33: 55796 Aborted (core dumped) ${RASCAF}/rascaf -b ${READS}/.._unfiltRNA.bam -f ${FASTA}/20190319.._001_001_1st_assembly_minION.ctn.fa -o ${TARGET}/rascaf_1st

SRR3453110.85720556 163 ctg1 18931 40 9M3I89M = 18980 150 ATTTATGATAAAAAAAAAGAATTTAATAGATGGGCACACTACTAGGCACACTTATTCAAAGTAAATAAGATTTAGGAGCATATATGTATGAATTTCAATTA ?@BFFFFDHHHFHIJJGGFFFGIJIGGDHIGGGGHHGHIJJIJIJJIJGGGCHHHHEEHBDBDFCCDEEECED>CCDD?BDDDEEECCFEDDEDCACCDDD AS:i:-24 XN:i:0 XM:i:2 XO:i:1 XG:i:3 NM:i:5 MD:Z:16C60C20 YS:i:-7 YT:Z:CP SRR3453110.89338447 163 ctg1 18940 42 101M = 18986 147 AAAAAAGAATTTAATAGATGGGCACACTACTAGGCACACTTATTCAAAGTAAATAAGATTTAGGAGCATATATGTATGAATTTCAATTAATTGAAACTGAG CCCFFFFFHHHHHJJJJJJJJJIJJJJJJJJJIJJJJJJJJJJJJJJJJGFHIJJJJIJJJJJJJJIGJHHHHHFHHFFFFFFFEEDEEDEEDDDDDCCCC AS:i:-11 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:7C60C32 YS:i:-5 YT:Z:CP SRR3453110.90544994 163 ctg1 18960 42 101M = 19116 256 GGCACACTACTAGGCACACTTATTCAAAGTAAATAAGATTTAGGAGCATATATGTATGAATTTCAATTAATTGAAACTGAGTTGGTATTTGGGTGATTTGT @@@DFDFFHFFBFEGGIJJHIEHIHFEE>HHGGIGDCBFHGEIB4?D?DB??BDGHEGCEHHHGC=BFGFDHHGFC@EGCGHGFFCE?CBDBB=AA;A@@> AS:i:-5 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:48C52 YS:i:-8 YT:Z:CP SRR3453110.89452103 99 ctg1 18970 42 101M = 19010 141 TAGGCACACTTATTCAAAGTAAATAAGATTTAGGAGCATATATGTATGAATTTCAATTAATTGAAACTGAGTTGGTATTTGGGTGATTTGTCAGGATTGAA @@CFFFDFHGFFHGGHIIIFHIGGJGIHIEHIGGGHIIIHIIIGBHGEGEFHGHGHIIJIIGGGICJEE>HCHIIIHGIIGF@;A=EBCDEFFFA;AEEDD AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:38C62 YS:i:-4 YT:Z:CP SRR3453110.85720556 83 ctg1 18980 40 101M = 18931 -150 TATTCAAAGTAAATAAGATTTAGGAGCATATATGTATGAATTTCAATTAATTGAAACTGAGTTTGTATTTGGGTGATTTGTCAGGATTGAAGAGACCAAAA @3;CCEDEDFFDDFCFHHHHFFGGDDGCHEGIEJGGBCDIHGIIIGEHDFBGEGGHGFDHGFC*GJJJIGHFHFGIJJJHDJJIHIGIHHHHHDDFFF@@C AS:i:-7 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:28C34G3YS:i:-24 YT:Z:CP SRR3453110.89338447 83 ctg1 18986 42 101M = 18940 -147 AAGTAAATAAGATTTAGGAGCATATATGTATGAATTTCAATTAATTGAAACTGAGTTGGTATTTGGGTGATTTGTCAGGATTGAAGAGACCAAAATGTCGC CCDDEDDDCCEDEFEEDFEGGHHFFGIJJIIIIJJJJIJJJJJIJJJJIIIIJJJJIJJJJJJJJJJJJJJJJJJJJJJJJJJJJIHIFHHHHFFFFFCCC AS:i:-5 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:22C78 YS:i:-11 YT:Z:CP SRR3453110.74320311 99 ctg1 19002 42 101M = 19034 132 GGAGCATATATGTATGAATTTCAATTAATTGAAACTGAGTTGGTATTTGGGTGATTTGTCAGGATTGAAGAGACCAAAATGTCGCTCAGTTTCGGCACCCT @@@DFFDDDHFHCEGHD>DHGEHEHIIBGFEFCHIGE?EB@C@::CDGFHA?68?D?BFHIIC>FGGII@GGII;=@6CH?7=2;/?ACCDEDB:?==?BA AS:i:-5 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:6C94 YS:i:-8 YT:Z:CP SRR3453110.89452103 147 ctg1 19010 42 101M = 18970 -141 TATGTATGAATTTCAATTAATTGAAACTGAGTTGGTATTTGGGTGATTTGTCAGGGTTGAAGAGACCAAAATGTCGCTCAGTTTCGGCACCCTGTTTCTGG DDCDDEDCDCCCCCEEDDCA>FDEFCDDCDDDECECCA?;FHHEHEEHGGHIHB@=GGGGGGIGHGIJJJIIIIGJIEFJIIIIJIHFAFGHFFFFDD@@@ AS:i:-4 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:55A45 YS:i:-6 YT:Z:CP SRR3453110.74320311 147 ctg1 19034 42 77M1I23M = 19002 -132 AACTGAGTTGGTATTTGGGTGATTTGTCAGGATTGAAGAGACCAAAATGTCGCTCAGTTTCGGCACCCTGTTTCTGGTTTTCGTCGAACATGGCAAACAAG >:CCCC@>CCCAAABBB?@>@>>@CCCC<AACCCAACCBBCDDCCD>A:>GGIHGGEGHGFB9IGBC@FIGGEGGBGDBGGGGFCCDFF??DDDBD??@ AS:i:-8 XN:i:0 XM:i:0 XO:i:1 XG:i:1 NM:i:1 MD:Z:100 YS:i:-5 YT:Z:CP SRR3453110.84141312 99 ctg1 19042 42 69M1I31M = 19116 174 TGGTATTTGGGTGATTTGTCAGGATTGAAGAGACCAAAATGTCGCTCAGTTTCGGCACCCTGTTTCTGGTTTTCGTCGAACATGGCAAACAAGAAGGTTTC B@CADDFFHHHFFHIJJJHIJJJIJIIJJIJJGIIJJJJIJHIJIJIFIIJIIIJJJIJJIHHHHHHFDBEFDECDBD??BDDCDDDDDDDDDCDDD?CDD AS:i:-8 XN:i:0 XM:i:0 XO:i:1 XG:i:1 NM:i:1 MD:Z:100 YS:i:-8 YT:Z:CP