Hi, I am a newbie learning to do SNP detection using exon reads of Chromosome 17 obtained from Sanger sequencing.
I have tried to use the GATK workflow:
BWA-0.7.12 (alignment)
PICARD-0.1.119 tools (sorting)
GATK-3.5 (calibration. realignemnt, SNP calling)
but only getting empty VCF file in the end.
I was unable to add the header for the bam files because I don't have any information for the read groups or library details
Is there any other softwares exist that can use short reads from Sanger to do SNP identification?
Hi Nithyanan,
Have you installed any program for sequence reader like Chromas from Technelysium (http://technelysium.com.au/?page_id=13) or Sequence scanner from ABI or DNA Baser, BioEdit, CLC, CodonCode aligner, Mutalyzer and so and so?. You just google it and will get plethora of softwares to read chromatogram (.ab1 / .seq) files. Just open your sequences using any of the platforms then BLAST it. Most of the sequence scanners has this in built option. Otherwise, just get your sequence in fasta format and Blast it in NCBI Blast. You may see the difference in homology and sequence pair between your sequence and reference sequence.
All the best,
Rajkumar
Hi Nithyanan,
The packages you mentioned are suitable for NGS reads. Whenever you work with Sanger sequencing data, just take it easy! Do the analysis in classic way and use a simple software like what Rajkumar mentioned.
Chromas and other app like it is more suitable when you are dealing with limited read number.
If You have a lots of read you may try NovoSNP app. It is free and also user-friendly.
Cheers,
Hamid
Hi Hamid,
Thanks for updating the discussion with new app.
Rajkumar
Hi Nithyanan,
May be this package "sangerseqR" from R be useful ,also may be you can use soap for SNPdetection.
Best,
Soheil
Better to follow the program for sequence reader like Chromas from Technelysium (http://technelysium.com.au/?page_id=13) or Sequence scanner from ABI or DNA Baser, BioEdit, CLC, CodonCode aligner, Mutalyzer, GeneRunner and sangerseqR you will get plethora of softwares to read chromatogram (.ab1 / .seq) files. Just open your sequences then BLAST it. Sequence scanners has this in built option so just get your sequence in fasta format and Blast it in NCBI Blast. Finally the difference in homology and sequence pair between your sequence and reference sequence be observed.
Best wishes
My suggestion when using scanners from ABI or DNA Baser you need install sequence editor such as BioEdit, or Codon Code aligner you can try to download the manual book known as geneious 9.0 by Biomatters LTD 2015 . form the manual you will encounter various input formats thereby you should chose the appropriate file format that fits the BLAST or FASTA platform. Afterwards you can search variation in homology and reference sequence within the short reads. for detection my advice is to try MAC and BWA or otherwise SOAP as my colleague suggested, but to my experience BWA prove to give better results.
Since you have short read sequences obtained by Sanger sequencing, just get clean sequences using classic software like Bioedit (you may manually revise each base for heterozygous with the help of the trace, i.e. your .ab1 file) and then do sequence assembly with SeqMan. You will detect every SNP (option Show SNPs) and get a SNP report.
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