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!