[MGCBrown]

Does anyone have the SNP_in_ORF_nonsyn.pl script that was described in this thread? The link to the script is no longer in use (http://users.ugent.be/~slvbelle/NGS/)

Any info would be great!

[maoshigua]

Hi Steven,
i send you those three input files. thanks a lot.

Maoshigua

[Steven VB]

Hi maoshigua,

can you send me ([email protected]) a sample of your data? I will try to fix it.

Cheers,
Steven

[maoshigua]

JackieBadger, I tried the script. It came with:
Error, Reference nucleotide does not equal the one in the original sequence at ./SNP_in_ORF_nonsyn_multiSNP.pl line 85, <GEN0> line 6.

any suggestions, please?

Maoshigua

[Steven VB]

JackieBadger, I tried the script. It came with:

Use of uninitialized value $countSyn in concatenation (.) or string at SNP_in_ORF_nonsyn.pl line 101, <GEN0> line 39393.
Use of uninitialized value $countNonSyn in concatenation (.) or string at SNP_in_ORF_nonsyn.pl line 102, <GEN0> line 39393.

any advice? pls.

Please check again: http://users.ugent.be/~slvbelle/NGS/

I made some modifications, it should work now...

[d1antho]

SNPdat

SNPdat can be used for this

http://www.biomedcentral.com/1471-2105/14/45

http://code.google.com/p/snpdat/

(there is also a short tutorial in the downloads section)

You only need a VCF for input, annotation file (GTF) and reference sequence (Fasta file). The annotation and sequence information can be from your own assembly and dont require any preprocessing.

[fanx]

JackieBadger, I tried the script. It came with:

Use of uninitialized value $countSyn in concatenation (.) or string at SNP_in_ORF_nonsyn.pl line 101, <GEN0> line 39393.
Use of uninitialized value $countNonSyn in concatenation (.) or string at SNP_in_ORF_nonsyn.pl line 102, <GEN0> line 39393.

any advice? pls.

[fanx]

Look at this pub. "De novo Transcriptome Assembly and SNP Discovery in the Wing Polymorphic Salt Marsh Beetle Pogonus chalceus (Coleoptera, Carabidae)"

I now provide a quote from the primary author, reference their paper if you use the script

"The script for finding amino acid changes uses several data files.

- I searched the ORFs in the unigenes with this program: http://proteomics.ysu.edu/tools/OrfPredictor.html

è Output: a CDS file (DNA sequences of the ORFs) and a PEP file (AA sequences of the ORFs, and also contains START, STOP and READINGFRAME of the ORFs)



- SNP calling with SAMtools

è Output: VCF file (SNP and positions of SNP)



- Perl script (SNP_in_ORF_nonsyn.pl) infers whether SNPs are located within an ORF and whether the SNP results in an amino acid change. The script gets the SNP position from the VCF file, mutates the position in the original sequence in the unigene fasta file, then translates that sequence according its ORF (from PEP file) and then checks whether the original sequence differs from the mutated sequence. The script uses bioperl.

è Output: each line in the VCF file that contains a nonsynonymous SNP. At the end, the number of synonymous and nonsynonymous is also outputted.



I made the script and data available here: http://users.ugent.be/~slvbelle/NGS/

(I added an example PEP and VCF file which should work)



The script should be used as follows:

./SNP_in_ORF_nonsyn.pl Trinity_GC018ALL_unique.fasta PEP.fasta SNP.vcf > output"

I haven’t tried it but I think your script on the end of the post should work (definitely cite that PLoS paper). I also wonder if there are alternative ways because my case is much simple. I sequenced a long and heterogeneous viral ORF using HiSeq 2000. Thus ORF prediction is unnecessary. My destination is to calculate the number of dS and dN over the viral ORF through a sliding window. Only tool I am aware is CLC’s SNP analysis tool from a publication (http://www.ncbi.nlm.nih.gov/pubmed/22278255). There may other facilities to be able to do this job too. Thanks in advance.

[JackieBadger]

Look at this pub. "De novo Transcriptome Assembly and SNP Discovery in the Wing Polymorphic Salt Marsh Beetle Pogonus chalceus (Coleoptera, Carabidae)"

I now provide a quote from the primary author, reference their paper if you use the script

"The script for finding amino acid changes uses several data files.

- I searched the ORFs in the unigenes with this program: http://proteomics.ysu.edu/tools/OrfPredictor.html

è Output: a CDS file (DNA sequences of the ORFs) and a PEP file (AA sequences of the ORFs, and also contains START, STOP and READINGFRAME of the ORFs)



- SNP calling with SAMtools

è Output: VCF file (SNP and positions of SNP)



- Perl script (SNP_in_ORF_nonsyn.pl) infers whether SNPs are located within an ORF and whether the SNP results in an amino acid change. The script gets the SNP position from the VCF file, mutates the position in the original sequence in the unigene fasta file, then translates that sequence according its ORF (from PEP file) and then checks whether the original sequence differs from the mutated sequence. The script uses bioperl.

è Output: each line in the VCF file that contains a nonsynonymous SNP. At the end, the number of synonymous and nonsynonymous is also outputted.



I made the script and data available here: http://users.ugent.be/~slvbelle/NGS/

(I added an example PEP and VCF file which should work)



The script should be used as follows:

./SNP_in_ORF_nonsyn.pl Trinity_GC018ALL_unique.fasta PEP.fasta SNP.vcf > output"

[fanx]

Hi

Anyone has any ideas how would one find out (programmatically) synonymous and non-synonymous snps from vcf files? I have used mpileup on several hundred bacterial genomes to get the vcf file.

Thanks

bioinfun, I have a similar problem. Are there any solutions now? Thanks.

[swbarnes2]

What I've done is using the coordiante from the vcf to get the sequence around and including the SNP. Then I blastx those sequences against a database of the proteins from that bacterium. Then I parse the blastx to find out which changes cause amino acid differences.

But yes, annovar is easier, if you can get a file for annovar to use to compare to.

[SeekAnswers]

You can try comparing the coordinates in the variant VCF with the coding region start/ends in refseq to see where your variant falls in and make a determination based on that.

[bioinfun]

Thanks guys but....

I am trying to program it myself and I thought I can get some leads into how to do this from a vcf file.

What do you think of this quick way of doing this:

1- get the nucleotide sequence of the CDS that has the SNP
2- perform 6-frame translation
3- compare with reference translated sequence
4- if the sequences are different then the SNP at point (1) is non-syn if they are the same then its syn.

Not accurate but will give you an idea. What do you guys think?

[iansealy]

Or Ensembl's VEP (http://www.ensembl.org/tools.html) or snpEff (http://snpeff.sourceforge.net/) or...

[ymc]

Well, you either write your own tool to do that or try annovar