Hmm, interesting! I don't think it's a "dye blob" since it's about the same height as your other peaks and the background is low. It might be due to an artifact in the capillary (capillary getting worn out), loading too much product, or it could be a real sequence.

You might need to clone and sequence your products to rule out this being biologically real and not an artifact. Plus, cloning will enable you to phase your SNPs. Or you could use a PacBio sequencing run.

I'd start by contacting the sequencing facility as see if it's possible that this is due to an issue with the age of the capillary tubes and/or loading too much product.

I think that it is probably a dye blob. The width and shape of the 3 dyes is not typical of sequence and although they are basecalled as Ts there is just too much material overlapping in too many colours to be normal sequence. On my sequencer if dye removal was incomplete due to poor sequencing ( so low incorporation of dye reagents) or poor separation of salts from sequence I got these wide peaks of unincorporated dye and the size of the peak was related to the size of the very small unincorporated dye blob at the start of the sequence. Can you attach an original .abi sequence file which may give some clues please?

Hi Galvin,

As Paul mentioned, what you see is probably a dye blob. You can investigate this by first of all checking the post-sequencing clean up process to see if for instance the right concentration of ethanol and salt has been used, etc. You may indeed want to use non-ethanol base protocols for the clean up process. Examining the raw sequence data to see if the sequencing reaction has failed would certainly be helpful.

Hope this helps,

Parham

Katie A Burnette ,

Thank you. I contacted the facility and they are confident it is a dye blob due to unincorporated fluorescent nucleotides ruining on the capillary. I am also not so sure about this as I sometimes have the "t" artifact in much smaller proportion.

Paul Rutland & Parham Habibzadeh ,

Thank you I appreciate the advice. I am fairly new to the laboratory and I am unsure how to go about checking the post-sequencing clean up processes. When I sent in my pcr product, I did ask for a clean-up of the product, but I am unaware of a post-sequencing clean up ( I assume this happen digitally to my sequences?)

I have attached .abi files.

1) 16-EXON2_FWD_OUT

This is the sequence with the large T artifact.

2) 30-EXON2_FWD_OUT

As you can see, same sequence but the T artifact has scaled down nearly 200%

Thank you again for all your input it has been a good learning experience.