This is a test version of Biostars. For the public version, visit https://www.biostars.org.
Ngs With Sanger

Hello all,

Our Sanger sequencing finds one homologous SNPs on chr7 for a patient with heart disease, but NGS data from three platforms (PGM, MiSeq and HiSeq) all show that site only has homologous of reference allele, how to explain this?

Many thanks

sequencing
• 3,397 views • link
updated by Bair • Bair • written by User 9166 • User 9166

Can you provide more information about the Sanger sequencing? How many sample repeats were performed? What does the chromatogram trace look like for the Sanger read?

• 0 views • link
written by Dan D Dan D

You probably mean homozygous, not homologous? In addition to looking at the sanger trace, I would also want to see the coverage for NGS that you have at this position.

• 0 views • link
written by Obi Griffith Obi Griffith

4 answers

Back in the past some cloning kits introduced mutations in cloned PCR fragments. Also "mutation calling" on Sanger PCR-fragment data can be nonsensical if there is high amplification background. show/send trace files to some old hats experienced with mutation screening.

• 0 views • link
written by Darked89 Darked89

What are the odds that it's a sample mix-up? These things do happen.

• 0 views • link
written by Swbarnes2 Swbarnes2

could it be misalignment? NGS reads are short and if there was a short stretch of sequence somewhere else in the genome that looked like your target region except that it was homo for the reference, the situation you described could have happened.

• 0 views • link
written by Wen.Huang Wen.Huang

Thank you all.

Several Sanger samples repeat the mutation, it's NGS coverages are high (>300) by each of the three platforms.

The length of reads is as long as 300bp, (100 in HiSeq, 150 in MiSeq and 20-300 in PGM), so it might be a sample mix-up problem.

Will let you informed ASAP.

• 1 views • link
written by Bair • Bair

Log in to answer this question.

More posts like this