Hello all!!!

I have sequenced the strain we have in the lab and now I want to use that assembly to make snp calling against other strains. The problem is while building the database with snpEff, I am always having this same error and I don't understand why :

java -jar snpEff.jar build -gff3 -v TB50

00:00:00 SnpEff version SnpEff 4.3t (build 2017-11-24 10:18), by Pablo Cingolani 00:00:00 Command: 'build' 00:00:00 Building database for 'TB50' 00:00:00 Reading configuration file 'snpEff.config'. Genome: 'TB50' 00:00:00 Reading config file: /Users/tmartinc/work/RL/PhD/Database/TB50seqLausanne/SNPs/snpEff/snpEff_latest_core/snpEff/snpEff.config 00:00:00 done Reading GFF3 data file : '/Users/tmartinc/work/RL/PhD/Database/TB50seqLausanne/SNPs/snpEff/snpEff_latest_core/snpEff/./data/TB50/genes.gff' Total: 0 markers added.

Create exons from CDS (if needed): 
Exons created for 0 transcripts.

Deleting redundant exons (if needed): 
    Total transcripts with deleted exons: 0

Collapsing zero length introns (if needed): 
    Total collapsed transcripts: 0
Reading sequences   :
FASTA file: '/Users/tmartinc/work/RL/PhD/Database/TB50seqLausanne/SNPs/snpEff/snpEff_latest_core/snpEff/./data/genomes/TB50.fa' not found.
Reading FASTA file: '/Users/tmartinc/work/RL/PhD/Database/TB50seqLausanne/SNPs/snpEff/snpEff_latest_core/snpEff/./data/TB50/sequences.fa'
    Reading sequence 'tb50_4_tig00000001_BK006938', length: 1527445
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_12_tig00000004_BK006945', length: 72496
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_15_tig00000013_BK006948', length: 1102713
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_16_tig00000014_BK006949', length: 941389
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_7_tig00000015_BK006941', length: 1091053
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_13_tig00000016_BK006946', length: 912520
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_2_tig00000018_BK006936', length: 832510
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_14_tig00000020_BK006947', length: 770263
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_10_tig00000022_BK006943', length: 744706
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_11_tig00000025_BK006944', length: 680926
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_8_tig00000027_BK006934', length: 576431
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_5_tig00000029_BK006939', length: 604804
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_9_tig00000033_BK006942', length: 425111
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_3_tig00000035_BK006937', length: 329289
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_6_tig00000037_BK006940', length: 266054
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_1_tig00000039_BK006935', length: 246943
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_Mito_tig00000041_KP263414', length: 84520
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_12_3_tig00000132_BK006945', length: 683993
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_12_2_tig00000133_BK006945', length: 88102
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
    Reading sequence 'tb50_12_1_tig00000134_BK006945', length: 482874
    Adding genomic sequences to exons:  Done (0 sequences added, 0 ignored).
Total: 0 sequences added, 0 sequences ignored.

Adjusting transcripts: 
Adjusting genes: 
Adjusting chromosomes lengths: 
Ranking exons: 
Create UTRs from CDS (if needed): 
Correcting exons based on frame information.

Remove empty chromosomes: 

Marking as 'coding' from CDS information: 
Done: 0 transcripts markedjava.lang.RuntimeException: FATAL ERROR: Most Exons do not have sequences!
Chromosome names missing in 'reference sequence' file:  , , , , , , , , , , , , , , , , , , , 
Chromosome names missing in 'genes' file             :  'tb50_10_tig00000022_BK006943''tb50_11_tig00000025_BK006944''tb50_12_1_tig00000134_BK006945''tb50_12_2_tig00000133_BK006945''tb50_12_3_tig00000132_BK006945''tb50_12_tig00000004_BK006945''tb50_13_tig00000016_BK006946''tb50_14_tig00000020_BK006947''tb50_15_tig00000013_BK006948''tb50_16_tig00000014_BK006949''tb50_1_tig00000039_BK006935''tb50_2_tig00000018_BK006936''tb50_3_tig00000035_BK006937''tb50_4_tig00000001_BK006938''tb50_5_tig00000029_BK006939''tb50_6_tig00000037_BK006940''tb50_7_tig00000015_BK006941''tb50_8_tig00000027_BK006934''tb50_9_tig00000033_BK006942''tb50_Mito_tig00000041_KP263414'

. File '/Users/tmartinc/work/RL/PhD/Database/TB50seqLausanne/SNPs/snpEff/snpEff_latest_core/snpEff/./data/TB50/genes.gff' line 13047 'tb50_Mito_tig00000041_KP263414 exonerate:protein2genome:local match_part 17983 18349 . + . Parent=match00344'

at org.snpeff.snpEffect.factory.SnpEffPredictorFactory.error(SnpEffPredictorFactory.java:421)
at org.snpeff.snpEffect.factory.SnpEffPredictorFactory.finishUp(SnpEffPredictorFactory.java:556)
at org.snpeff.snpEffect.factory.SnpEffPredictorFactoryGff.create(SnpEffPredictorFactoryGff.java:348)
at org.snpeff.snpEffect.commandLine.SnpEffCmdBuild.run(SnpEffCmdBuild.java:369)
at org.snpeff.SnpEff.run(SnpEff.java:1183)
at org.snpeff.SnpEff.main(SnpEff.java:162)

java.lang.RuntimeException: Error reading file '/Users/tmartinc/work/RL/PhD/Database/TB50seqLausanne/SNPs/snpEff/snpEff_latest_core/snpEff/./data/TB50/genes.gff' java.lang.RuntimeException: FATAL ERROR: Most Exons do not have sequences! Chromosome names missing in 'reference sequence' file: , , , , , , , , , , , , , , , , , , , Chromosome names missing in 'genes' file : 'tb50_10_tig00000022_BK006943''tb50_11_tig00000025_BK006944''tb50_12_1_tig00000134_BK006945''tb50_12_2_tig00000133_BK006945''tb50_12_3_tig00000132_BK006945''tb50_12_tig00000004_BK006945''tb50_13_tig00000016_BK006946''tb50_14_tig00000020_BK006947''tb50_15_tig00000013_BK006948''tb50_16_tig00000014_BK006949''tb50_1_tig00000039_BK006935''tb50_2_tig00000018_BK006936''tb50_3_tig00000035_BK006937''tb50_4_tig00000001_BK006938''tb50_5_tig00000029_BK006939''tb50_6_tig00000037_BK006940''tb50_7_tig00000015_BK006941''tb50_8_tig00000027_BK006934''tb50_9_tig00000033_BK006942''tb50_Mito_tig00000041_KP263414'

. File '/Users/tmartinc/work/RL/PhD/Database/TB50seqLausanne/SNPs/snpEff/snpEff_latest_core/snpEff/./data/TB50/genes.gff' line 13047 'tb50_Mito_tig00000041_KP263414 exonerate:protein2genome:local match_part 17983 18349 . + . Parent=match00344'

at org.snpeff.snpEffect.factory.SnpEffPredictorFactoryGff.create(SnpEffPredictorFactoryGff.java:353)
at org.snpeff.snpEffect.commandLine.SnpEffCmdBuild.run(SnpEffCmdBuild.java:369)
at org.snpeff.SnpEff.run(SnpEff.java:1183)
at org.snpeff.SnpEff.main(SnpEff.java:162)

My sequences.fa has headers like this:

>tb50_4_tig00000001_BK006938
>tb50_12_tig00000004_BK006945

And my genes.gff looks like this:

tb50_4_tig00000001_BK006938 exonerate:protein2genome:local  match   771461  771859  718 +   . ID=match03090;Name=sp|A0A023PZE8|YD57W_YEAST;Target=sp|A0A023PZE8|YD57W_YEAST 1 133;Gap=M399
tb50_4_tig00000001_BK006938 exonerate:protein2genome:local  match_part  771461  771859  .   +   .   Parent=match03090

So for me, the headers are correct and same in both files... Where am I wrong???

I also checked this error in the snpEff website (http://snpeff.sourceforge.net/SnpEff_manual.html#trouble) and I followed the intructions to add the sequences to the gff3 file but still same error...

Please help!

Thanks a lot!!! Trini