I am trying to sequence thru the trnH-psbA intergenic spacer region in my plant taxa. But, it has homopolymer/mononucleotide runs of 10 or more T's or A's. I get amplification products, but get really bad sequence reads downstream of these mononucleotide repeats of >10 Thymines or Adenines. I'm stuck! Anyone have any suggestions???
@Artur,
I tried multiple rounds of fwd and rev sequencing, at various primer and PCR product concentrations, and I still have difficulty sequencing through regions from both ends--thus making it really difficult to assemble contigs :(
If knowing the length of the homopolymer is not important and you have a reference sequence, this can be overcome. The trick is to place the primers on the edges of the region pointing out, creating two amplicons. With the primer 3' landing off the end of the homopolymer region as needed for specificity. Also, avoid using pyrosequencing when sequencing homopolymer regions. If you have no reference sequence or good-guesses or need to know the length of the homopolymer, there are other options involving NGS and custom informatics.
Hi Kuahi,
I disagree with Artur in that the 454 and Ion Torrent platforms are very unreliable for sequencing homopolymer runs greater than 5 or 6bp, while Sanger-based and Illumina platforms are reasonably reliable (although you might have a look here: http://omicsomics.blogspot.com/2012/01/does-illlmina-also-have-homopolymer.html). The reason has to do with whether the platform utilizes blocking moieties attached to added nucleotides (Sanger uses chain-terminating di-deoxynucleotides, Illumina uses reversible terminators, 454 and Ion use neither).
With that said, it sounds like your problem is more likely with your amplification rather than your sequencer. I would suggest testing with as many alternative primer combinations as you can, and also checking all primer sequences to see whether they might be annealing elsewhere in the plastid genome. If you are using Sanger, you might also have a look at some of the available troubleshooting guides like this one:
https://tools.thermofisher.com/content/sfs/manuals/MAN0014435_Trbleshoot_Sanger_seq_data_UB.pdf?cid=techcomm92
Hope this helps, and good luck!
Matt
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