rascaf_error

[r1corre]

Dear developper I'm trying to use rascaf on my genome with the following command rascaf -b /scratch/fr2424/sib/corre/my.bam -o ind2_0 -f /scratch/fr2424/sib/corre/mygenome.fasta -o /scratch/fr2424/sib/corre/rascaf_out

My job end with the following error

Found 232107 exon blocks. rascaf: blocks.hpp:554: void Blocks::GetAlignmentsInfo(Alignments&): Assertion `readCnt > 30' failed. rascaf failed.

and this output file

head rascaf_out.out

Contigs 0: opera_scaffold_35 1 21160 1: opera_scaffold_35 21162 30042 2: opera_scaffold_35 30044 44257 3: opera_scaffold_35 44259 49471 4: opera_scaffold_35 49473 51592 5: opera_scaffold_35 51594 54391 6: opera_scaffold_35 54393 56418 7: opera_scaffold_35 56420 65899 8: opera_scaffold_35 65901 67093 9: opera_scaffold_35 67095 68230 10: opera_scaffold_35 68232 69045 11: opera_scaffold_35 69047 85263 12: opera_scaffold_35 85265 91369 13: opera_scaffold_35 91371 95685 14: opera_scaffold_35 95687 106381 15: opera_scaffold_35 106383 106886 16: opera_scaffold_35 106888 107071 17: opera_scaffold_35 107073 111205 18: opera_scaffold_35 111207 136990 19: opera_scaffold_35 136992 144728 20: opera_scaffold_35 144730 147483

Do you have any ideas about how to solve this error?

Regards Erwan

[mourisl]

Could you please show me the first few alignments from the bam file (like ~20?).

[r1corre]

Hi Here are the firsts alignments RNAseq reads have been align on the genome using hisat

Thanks for your help @SQ SN:C14250269 LN:100 @PG ID:hisat2 PN:hisat2 VN:2.0.5 CL:"/usr/local/genome2/hisat/hisat2-align-s --wrapper basic-0 -x ../hisat_indx/a.pompejana -S ind2.sam -p 16 -1 /projet/sbr/abice-rnaseq/Alvinellidae/genome_AP/ind02/HI.3608.005.Inde x_21.2_R1.fastq -2 /projet/sbr/abice-rnaseq/Alvinellidae/genome_AP/ind02/HI.3608.005.Index_21.2_R2.fastq" HISEQ:366:C90UCANXX:5:1101:1362:1965 77 0 0 0 0 GNTCAACCAGGTAACCAACCAAAACGCTCGGTCACTGACACTGCTGCCCGCAACACCCATCCAACCACCTACAAGCAACGCTACCGGCCTGCCGACCACAGTGCCAACAACCAAGCCTTTCCAAC <#<//B BFBFFBFFFFFFFFFFFFFFBFFFFFF<FFFFFFFFFFFFF/FF/BFFFFFFFFFBBFFFFF<F/BF<FFBFFFF//B/FFFFFFFFFFBBBFFFFFFFF</7//B/BBFF<//B/7<F YT:Z:UP HISEQ:366:C90UCANXX:5:1101:1362:1965 141 0 0 0 0 TCTAGGCACTGATTCCAAAATATCAGTGGTGGGCTCTGCAGCTTGTGAGGCCTGGTGCTATCGGAGAGAGCCAGGCAAGTTGGACGCCTGGGTTTGTCTTGAGGTAGTGGTAGGGCCGGCCGCGT </BBB/ /FFFFF/F<FBFFFB<FFFFBBBFF/B/<FF/FFFBFF/F//</<B<FBF//F/<FBF7B//F</</BB//<FFF/BBB//<BFBF/FF/FFBB<BBB7B/<BFFFFFFF</<B##### YT:Z:UP HISEQ:366:C90UCANXX:5:1101:1391:1982 77 0 0 0 0 CTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTCACCCTAGCCCTAACCCTAACCCTAACCCTAACCCTACCCCTAACACTAGC BB/BB< FB/FFFFFF/BFB<//<BBFF/<B/<F/</<<F/<BFFB/</<B<BFF/</FF</B/</<<//</////<<F/<F/<BFFB//<BBFF/<B/7B/7FB##################### YT:Z:UP HISEQ:366:C90UCANXX:5:1101:1391:1982 141 0 0 0 0 TTAGGGTTAGGGGTAGGGTTAGGGTTAGGGTTAGGGTTAGGGGTAGGGGTGGGGTGAGGGGTGGGGTTGGGGTTAGGGTTAGGGATAGGGTTAGGGTTAGGGGTCGGGTTAGTGGTAGGGGTGGG <<</<F B/FFBF/<<<FF<<<B/F/<BFF/FFFFBF/<</B</<FFBF////7<F/<B/<<B/7B############################################################ YT:Z:UP HISEQ:366:C90UCANXX:5:1101:1603:1982 153 C15664228 50 60 125M = 50 0 AAGACCTTTGACCCTAAACATTTGTCTATTACATTAGATACCAAAATAAACCGTGTTCAGTATTGTATACACAATGTCCTGCCATTACTTGTGACACCTTGAAAGATAAGGTTTTAGTATTTCTTFFFBFFFFFFBFBBFFFFF/FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBBB AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YT:Z:UP NH:i:1 HISEQ:366:C90UCANXX:5:1101:1603:1982 69 C15664228 50 0 = 50 0 TGAAATGAAGCTGAATGGAAGCTGAATGGAAGGTGAATGAAGCTGAACGAAGCTGAACGAAGCTGAAATGAAGCTGAAATGAAGCTGAATAAAAGCTGAATAGAAGCGTGCAATTCCAAAGAAGCBBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBFFFFFFFFF YT:Z:UP HISEQ:366:C90UCANXX:5:1101:2516:1974 77 0 0 0 0 TTTGTCACGTTCGTTCAGATGAAGTTTACTTTGGGCCAACATAACGCAGCTGTTTATAGTCAACGCAAGATTGCTTTGACTTGTCTTCTCAAGTTCGGTGGTTTATACTTATAAAAAAGGACGTT BBBBBF FFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFF<F<BFFFFFBFF/FBFFFFFFFBBFFFFFFF<FFFFFFFFFFFFFFBFBFFFB<BF/FFFFFFBFF7FFFFBFFFBBF<FFF</BFF YT:Z:UP HISEQ:366:C90UCANXX:5:1101:2516:1974 141 0 0 0 0 GGAGAACTGGAGAATCGTCCAAGCAGCAGTCAGCGTCACCATAAGCAAGCACGTTGCAAACAAAGTTCTCCGCATCTTTGTTTTCTTTAACTCACTCCGTTCTGTTAACACGACATTACTCACCC BBBBBF FFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP HISEQ:366:C90UCANXX:5:1101:2129:1993 77 0 0 0 0 GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTTCGGAATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAACAATCACCAGCGCACGCAGACGACATACAAAACAGAAACAAGCG BBBBBF FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF/<BFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF################################################## YT:Z:UP HISEQ:366:C90UCANXX:5:1101:2129:1993 141 0 0 0 0 GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTGGGTCTCGGTGGTGGGCGGAGCGGTAAAAAAAAAAAAAAAAAAAGAAAGGAGGGGAGGAGCGGGGGAAGTAGGAGGGAAGAAGGGCGAGTAGA BBBBBF /F<<<<B<FB7B/BFFF<BFBFB################################################################################################ YT:Z:UP HISEQ:366:C90UCANXX:5:1101:1792:1995 99 opera_scaffold_545 700 60 62M2I61M = 922 347 TGCCGACAGCCGGTCGGACGGGTTCGTTCCGGCTGCTATGTTTTTATGGGCGGCCACCCACACGCGCGCGCGCGCGCGCGCGCGGACCGAGTCTGTCGGAGCTCGGCGTA TCTCTCGATCGGCGG BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFB/7<BFBFFFF/FBFFFF/B/BBB<//BFFFFF</B/7BFB AS:i:-11 XN:i:0 XM:i:0 XO:i:1 XG:i:2 NM:i:2 MD:Z:123 YS:i:-10 YT:Z:C P NH:i:1 HISEQ:366:C90UCANXX:5:1101:1792:1995 147 opera_scaffold_545 922 60 125M = 700 -347 CGACATCTCCTACGCCAAACACTCAGCACGTCTCCTCAAAACGCACGTACACAAGCACGTACGCAGGACACCCCTGGACACAGGTGACACAACAGGATGGCAATGAGTAAGTTTGCTT ATGATTT BFBFFFFFFFFFFBFFFFFFFFFFFFFFFF/FBFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBBB AS:i:-10 ZS:i:-11 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:9G0C114 YS:i:-11 YT:Z:CP NH:i:1 HISEQ:366:C90UCANXX:5:1101:2547:1973 83 opera_scaffold_237 15880 60 125M = 15667 -338 AGAAAAAGGCTTAACGTCTCCGTGATGAGTCCATCACTGAATGGAATATAAAAAGGACATTTATGGCAGAATTAATATAGGAATATTATAGTGGTTGGGTTCCCTCACACAGACACAC ACAGATT B/<FBFFFBBF<FFFF<<B<FFFFFFFFF</FBFFFFFFFFFFFFFFFFFFFFFFFFFFF/FFBFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFBFFFFFFBBBBBB AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP NH:i:1 HISEQ:366:C90UCANXX:5:1101:2547:1973 163 opera_scaffold_237 15667 60 125M = 15880 338 AACCAACCAACCAACCCACTCTCAACTATCTGCCTAACCTATCGTTCAGCACACAAACGTTGCCTTGATCGACTGATCAATCGATCGATCTTTATCAATCTTTATCTACCTCCTCTAT AACTGGA BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFF# AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP NH:i:1 HISEQ:366:C90UCANXX:5:1101:2532:1985 83 opera_scaffold_2861 7057 60 125M = 6854 -328 TTAATTGATGTTATAAAATCTATTGTTCCTCTGCTTTTAACAAAGAAATTGGAAAACCATTAACTCTGAAATGTGTCTATTGTGCTTTTGATTGGTGCATTAGCATGATTACCAGATG TTTGTTA FB7FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBBB AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP NH:i:1 HISEQ:366:C90UCANXX:5:1101:2532:1985 163 opera_scaffold_2861 6854 60 125M = 7057 328 CTGTTTTGTTTATCATAGGATGAAGAGGATGAGGAAGAAGAAGAAGAATCATCAAAGGAACCAGAAATACTTGGCCATGGAGAGAGAGCTGCTAGAGCCATTCTACAGAACAGAACAA GGGTTAG BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP NH:i:1 HISEQ:366:C90UCANXX:5:1101:3719:1987 73 C14520215 1 60 13S112M = 1 0 GCCGGACAGTGAAGAATTCTTAGAAGACGATCTCCCCGAAACCTGCGCTTCATTCAGTACCAACGCACAACAAAGAACGACGTCAATTACAAGATTTCGCTTTGAGATTCTAGGCAAATATGTTC</</</BBBB/<B/FBBFBFF//F/BF//<B/F/<B/<<<7<<<BF<FFF/<FBF<///<<//<B7/77/7FF///<B//7/7B//7/B//<</7FBFF<FFFFB/77B/7//////7F7BBF## AS:i:-17 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:52A59 YT:Z:UP NH:i:1 HISEQ:366:C90UCANXX:5:1101:3719:1987 133 C14520215 1 0 = 1 0 ATGCCCCTCTAGGAGCCTTAATGACCCGTTTTTCTTCATTAACATTAAATAAGCACCTTCCAACCATACTAATAGATATTTTTCTGGTCACCATATAACAACTAAGCCAATGGCATACGTTGCAC############################################################################################################################# YT:Z:UP HISEQ:366:C90UCANXX:5:1101:3946:1968 99 opera_scaffold_974 30227 60 125M = 30431 329 GNTACACGTGCAGCTCTACCGACGGTCAGCGGAAGTATCTTCTCCCGGAGGCTACAGACACTTGTCATCTAGAAACGGATAGCCAACAAGCCAGCAAACCGATCGAAAATCACAACTT TACCGAG /#<<//BFFFFFFF<F<FFFF<BB/BF<BFFBFFFF<<FFFFFFFBFFFFF/BFFFFFFFFFFFFFFFFFB<<7FFFFFFFF/7F<7B7FFFBFB/FFFFF<FFB/B<FFFFFBFB<FF</<FF7 AS:i:-2 ZS:i:-2 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:1A123 YS:i:-2 YT:Z:CP NH:i:1

[mourisl]

It seems the bam file is not sorted by coordinate. Can you use "samtools sort" to create a sorted bam file and try again? Thanks.