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Duplication of ITS-5.8s Amplicon (following Sanger sequencing and nBLAST) - (Jul/24/2018 )



I'm identifying various yeast isolates (different species) by sequencing the ITS1-5.8s-ITS2 region. After inputting some of the sequences through nBLAST, I started noticing that BLAST was using the same subject sequence, containing the full ITS region, twice within my query sequence. I have attached a picture for clarification.


Attached Image

Range 1 (bp 62 to 397 of subject sequence) contains part of ITS1 and the full 5.8s-ITS2 region, while Range 2 (36 to 350) contains the full ITS1-5.8s-ITS2 region. My full query sequence shown in the alignment goes from bp 16 (in Range 1) to bp 661 (in Range 2).


I cannot figure out how a second full ITS region could follow a previous ITS region since the full tandem repeat also includes regions on either side of the ITS region (18s and 28s). I have not been successful in finding anything about this issue in literature. I do plan to redo the sequencing, but since several of the isolates had a similar issue, I wanted to see if anyone might know the cause.


Sanger sequencing was performed using the forward primer ITS1 (TCCGTAGGTGAACCTGCGG). The reverse primer (ITS4-TCCTCCGCTTATTGATATGC) complementary sequence does appear near the end of range 1. So I have considered maybe it is a PCR issue where the ITS4 primer I used was defective, but that still would not explain why there are two fairly similar ITS regions one after another.


ANY help you can provide is greatly appreciated!


Thank you,



You've done well to include lots of information to help us help you. A few more questions, though: you said "several of the isolates had a similar issue." Does that mean you had other isolates that had the BLAST results you expected (one range)? if so, were all of them (odd and normal) amplified with the same primers (same batch)?  And was the PCR fragment size what you expected on a gel (is 841 bp the correct expected size?) for all the isolates (odd ones and normal ones?)  Were the chromatograms normal looking? or was there double sequence by any chance?


This is Old Cloner again- a few more questions: Did you assemble the two sequences produced by sequencing with ITS1 and ITS4 with some kind of software before submitting it to BLAST? How big was the assembled sequence? Was it the size you expected?


Never mind the first question above- I just realized you said you sequenced with only the ITS1 primer.  So it appears you got about 660 bp+ readable sequence, and the ITS4 binding site was in the middle (at about 329-358 bases of the part of the chromatogram that you submitted). That is very weird.  How big was the amplified band on a gel? Did the chromatogram go to the end of the fragment or become unreadable before it got that far? The questions in my above post are still important. Also you might try sequencing with the ITS4 primer and see if you get double sequence with that.


Sorry to keep adding edits- but this is really interesting! Did you get a single PCR product (one band) or more than one? By any chance did you gel-purify it before you sequenced it?


Do you have a control organism, an ATCC strain of S. cerevisiae for instance, as a control? And if so, did it give the sequence you expected after using these primers to amplify the sequence?