Issue in creating Database using ANNOVAR

[BiodeB]

Dear Sir,

I'm first time using this software for annotation of SNPs of Covid genomes. First I started creating a database following (https://github.com/WGLab/Workshop_Annotation and https://annovar.openbioinformatics.org/en/latest/user-guide/gene/#what-about-gff3-file-for-new-species) documentation of ANNOVAR. I've used reference genome of SARS COV-2 (NC_045512.2), and downloaded gtf file from ENSEMBL (https://covid-19.ensembl.org/Sars_cov_2/Info/Index).

Commands are

./gtfToGenePred -genePredExt Sars_cov_2.ASM985889v3.101.gtf Cov19_refGene3.txt

perl /home/imdeb/Softs/annovar/retrieve_seq_from_fasta.pl --format refGene --seqfile Cov19_Ref_NC_045512.2.fasta Cov19_refGene3.txt --out Cov19_refGene.fa

It gives me following message and Cov19_refGene.fa is empty

NOTICE: Reading region file Cov19_refGene3.txt ... Done with 12 regions from 1 chromosomes WARNING: Unable to retrieve regions at MN908947.3 due to lack of sequence information WARNING: Cannot identify sequence for ENSSAST00005000002 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000003 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000004 (starting from MN908947.3:21562) WARNING: Cannot identify sequence for ENSSAST00005000006 (starting from MN908947.3:25392) WARNING: Cannot identify sequence for ENSSAST00005000010 (starting from MN908947.3:26244) WARNING: Cannot identify sequence for ENSSAST00005000007 (starting from MN908947.3:26522) WARNING: Cannot identify sequence for ENSSAST00005000011 (starting from MN908947.3:27201) WARNING: Cannot identify sequence for ENSSAST00005000009 (starting from MN908947.3:27393) WARNING: Cannot identify sequence for ENSSAST00005000012 (starting from MN908947.3:27755) WARNING: Cannot identify sequence for ENSSAST00005000008 (starting from MN908947.3:27893) WARNING: Cannot identify sequence for ENSSAST00005000005 (starting from MN908947.3:28273) WARNING: Cannot identify sequence for ENSSAST00005000013 (starting from MN908947.3:29557) NOTICE: Finished writting FASTA for 0 genomic regions to Cov19_refGene.fa

So, this is a humble request that please furnish some valuable suggestion to overcome this issue.

[BiodeB]

The Problem might have solved.

Thank you

[kaichop]

Thank you.

[kaichop]

The warning message basically says that the MN908947.3 is not found in the input file (the fasta file). Are you sure it is supposed to be MN908947 rather than NM908947? If you want to do sars-cov-2 annotation, you could just use the sars-cov-2 reference file that I already prepared within the ANNOVAR package directly, because it is not straightforward for a new user to create such a file for viral genome which has complications such as ribosome slippage and pre-protein and so on. I already did the preparation so you can just use what I created for annotation.

…

On Sat, May 14, 2022 at 9:32 AM Debajyoti Kabiraj ***@***.***> wrote: Dear Sir, I'm first time using this software for annotation of SNPs of Covid genomes. First I started creating a database following ( https://github.com/WGLab/Workshop_Annotation and https://annovar.openbioinformatics.org/en/latest/user-guide/gene/#what-about-gff3-file-for-new-species) documentation of ANNOVAR. I've used reference genome of SARS COV-2 (NC_045512.2), and downloaded gtf file from ENSEMBL ( https://covid-19.ensembl.org/Sars_cov_2/Info/Index). Commands are ./gtfToGenePred -genePredExt Sars_cov_2.ASM985889v3.101.gtf Cov19_refGene3.txt perl /home/imdeb/Softs/annovar/retrieve_seq_from_fasta.pl --format refGene --seqfile Cov19_Ref_NC_045512.2.fasta Cov19_refGene3.txt --out Cov19_refGene.fa It gives me following message and Cov19_refGene.fa is empty NOTICE: Reading region file Cov19_refGene3.txt ... Done with 12 regions from 1 chromosomes WARNING: Unable to retrieve regions at MN908947.3 due to lack of sequence information WARNING: Cannot identify sequence for ENSSAST00005000002 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000003 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000004 (starting from MN908947.3:21562) WARNING: Cannot identify sequence for ENSSAST00005000006 (starting from MN908947.3:25392) WARNING: Cannot identify sequence for ENSSAST00005000010 (starting from MN908947.3:26244) WARNING: Cannot identify sequence for ENSSAST00005000007 (starting from MN908947.3:26522) WARNING: Cannot identify sequence for ENSSAST00005000011 (starting from MN908947.3:27201) WARNING: Cannot identify sequence for ENSSAST00005000009 (starting from MN908947.3:27393) WARNING: Cannot identify sequence for ENSSAST00005000012 (starting from MN908947.3:27755) WARNING: Cannot identify sequence for ENSSAST00005000008 (starting from MN908947.3:27893) WARNING: Cannot identify sequence for ENSSAST00005000005 (starting from MN908947.3:28273) WARNING: Cannot identify sequence for ENSSAST00005000013 (starting from MN908947.3:29557) NOTICE: Finished writting FASTA for 0 genomic regions to Cov19_refGene.fa So, this is a humble request that please furnish some valuable suggestion to overcome this issue. — Reply to this email directly, view it on GitHub <#192>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABNG3OFBUSWY4ADTNE3IAKTVJ6TQBANCNFSM5V5SO3RQ> . You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[gcha31]

Hi @kaichop
I am having issues during the preparation of annovar database with the SARS-CoV-2 genome. May I ask how can I access the reference file which you prepared for this purpose? Thanks a lot.
Best,
gc

[kaichop]

It is already included in the ANNOVAR package and you can directly use it.

…

On Tue, Jun 14, 2022 at 2:24 AM gcha31 ***@***.***> wrote: Hi @kaichop <https://github.com/kaichop> I am having issues during the preparation of annovar database with the SARS-CoV-2 genome. May I ask how can I access the reference file which you prepared for this purpose? Thanks a lot. Best, gc — Reply to this email directly, view it on GitHub <#192 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABNG3OAFUQNYBJCNAHNJJLLVPAQTBANCNFSM5V5SO3RQ> . You are receiving this because you were mentioned.Message ID: ***@***.***>

[tomswade]

Dear Sir,

I'm first time using this software for annotation of SNPs of Covid genomes. First I started creating a database following (https://github.com/WGLab/Workshop_Annotation and https://annovar.openbioinformatics.org/en/latest/user-guide/gene/#what-about-gff3-file-for-new-species) documentation of ANNOVAR. I've used reference genome of SARS COV-2 (NC_045512.2), and downloaded gtf file from ENSEMBL (https://covid-19.ensembl.org/Sars_cov_2/Info/Index).

Commands are

./gtfToGenePred -genePredExt Sars_cov_2.ASM985889v3.101.gtf Cov19_refGene3.txt

perl /home/imdeb/Softs/annovar/retrieve_seq_from_fasta.pl --format refGene --seqfile Cov19_Ref_NC_045512.2.fasta Cov19_refGene3.txt --out Cov19_refGene.fa

It gives me following message and Cov19_refGene.fa is empty

NOTICE: Reading region file Cov19_refGene3.txt ... Done with 12 regions from 1 chromosomes WARNING: Unable to retrieve regions at MN908947.3 due to lack of sequence information WARNING: Cannot identify sequence for ENSSAST00005000002 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000003 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000004 (starting from MN908947.3:21562) WARNING: Cannot identify sequence for ENSSAST00005000006 (starting from MN908947.3:25392) WARNING: Cannot identify sequence for ENSSAST00005000010 (starting from MN908947.3:26244) WARNING: Cannot identify sequence for ENSSAST00005000007 (starting from MN908947.3:26522) WARNING: Cannot identify sequence for ENSSAST00005000011 (starting from MN908947.3:27201) WARNING: Cannot identify sequence for ENSSAST00005000009 (starting from MN908947.3:27393) WARNING: Cannot identify sequence for ENSSAST00005000012 (starting from MN908947.3:27755) WARNING: Cannot identify sequence for ENSSAST00005000008 (starting from MN908947.3:27893) WARNING: Cannot identify sequence for ENSSAST00005000005 (starting from MN908947.3:28273) WARNING: Cannot identify sequence for ENSSAST00005000013 (starting from MN908947.3:29557) NOTICE: Finished writting FASTA for 0 genomic regions to Cov19_refGene.fa

So, this is a humble request that please furnish some valuable suggestion to overcome this issue.

@BiodeB
now I have encountered the same problem as you, may I ask how you solved it then? If you can reply me in your busy schedule, thank you very much!

[kaichop]

The covid genome and annotation are already included in annovar package and you do not need to create one yourself. Just directly use the annovar package. perl table_annovar.pl example/ex3.avinput sarscov2db -build NC_045512v2 -protocol avGene -operation g -out ex3 If you used a non-standard reference sequence to generate variant calls, then it is necessary to build it yourself (but in this case, you have to generate your own gene prediction as well).

…

On Mon, May 6, 2024 at 9:30 AM tomswade ***@***.***> wrote: Dear Sir, I'm first time using this software for annotation of SNPs of Covid genomes. First I started creating a database following ( https://github.com/WGLab/Workshop_Annotation and https://annovar.openbioinformatics.org/en/latest/user-guide/gene/#what-about-gff3-file-for-new-species) documentation of ANNOVAR. I've used reference genome of SARS COV-2 (NC_045512.2), and downloaded gtf file from ENSEMBL ( https://covid-19.ensembl.org/Sars_cov_2/Info/Index). Commands are ./gtfToGenePred -genePredExt Sars_cov_2.ASM985889v3.101.gtf Cov19_refGene3.txt perl /home/imdeb/Softs/annovar/retrieve_seq_from_fasta.pl --format refGene --seqfile Cov19_Ref_NC_045512.2.fasta Cov19_refGene3.txt --out Cov19_refGene.fa It gives me following message and Cov19_refGene.fa is empty NOTICE: Reading region file Cov19_refGene3.txt ... Done with 12 regions from 1 chromosomes WARNING: Unable to retrieve regions at MN908947.3 due to lack of sequence information WARNING: Cannot identify sequence for ENSSAST00005000002 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000003 (starting from MN908947.3:265) WARNING: Cannot identify sequence for ENSSAST00005000004 (starting from MN908947.3:21562) WARNING: Cannot identify sequence for ENSSAST00005000006 (starting from MN908947.3:25392) WARNING: Cannot identify sequence for ENSSAST00005000010 (starting from MN908947.3:26244) WARNING: Cannot identify sequence for ENSSAST00005000007 (starting from MN908947.3:26522) WARNING: Cannot identify sequence for ENSSAST00005000011 (starting from MN908947.3:27201) WARNING: Cannot identify sequence for ENSSAST00005000009 (starting from MN908947.3:27393) WARNING: Cannot identify sequence for ENSSAST00005000012 (starting from MN908947.3:27755) WARNING: Cannot identify sequence for ENSSAST00005000008 (starting from MN908947.3:27893) WARNING: Cannot identify sequence for ENSSAST00005000005 (starting from MN908947.3:28273) WARNING: Cannot identify sequence for ENSSAST00005000013 (starting from MN908947.3:29557) NOTICE: Finished writting FASTA for 0 genomic regions to Cov19_refGene.fa So, this is a humble request that please furnish some valuable suggestion to overcome this issue. @BiodeB <https://github.com/BiodeB> now I have encountered the same problem as you, may I ask how you solved it then? If you can reply me in your busy schedule, thank you very much! — Reply to this email directly, view it on GitHub <#192 (comment)>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABNG3OGS7B377K73Y6UQSMDZA6AWPAVCNFSM5V5SO3R2U5DIOJSWCZC7NNSXTN2JONZXKZKDN5WW2ZLOOQ5TEMBZGYYDENJZHE4Q> . You are receiving this because you were mentioned.Message ID: ***@***.***>