Hello,
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.
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,
Carmen