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sequencing result from excised DGGE bands

DGGE excised bands sequencing

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3 replies to this topic

#1 chin

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Posted 07 March 2012 - 04:03 AM

Dear all,

i'm currently doing soil microbe analysis using DGGE, i would like to seek for anyone who doing sequencing from excised dgge bands.

There are a lot of different species and genus from the blast result. I'm using NCBI blast after i get my sequence. How to choose the suitable species or genus from the blast result?. As my PCR products with only about 470bp.

Some of my samples from the blast result can give me up to 10 different species and all with Query coverage and Max ident at 99%.

Thank you.

#2 Trof

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Posted 07 March 2012 - 04:29 AM

What sequence do you DGGE? Some kind of ribosomal gene?
You need to look for 100% coverage and identity. Some of the mismatches may be basecalling errors, and some polymorphisms or mutations. You need to check the sequence manualy and see.
If you want to identify species from another, the species must differ in your sequence. Look for the species with the highest score, that is the closest match. If the score is the same, maybe that 10 BLASTed species have identical sequence. In that case you can't distinguish them.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

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#3 chin

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Posted 07 March 2012 - 05:18 AM

Appreciate for your reply.

i'm doing 18s soil algae DNA. The best i get only up to 99% for most of my sequenced results. I do check manually the sequences, yet there are at lease 1 or 2 mismatch(s).
As the PCR products only have about 470bp for the ease of DGGE, it seem like almost impossible to distinguish and confirm the sequences i get. So i wonder is it possible i set a bottom lines (maybe to 97% of Max Ident) to 'accept' all the BLASTed species? is it any other better way to interpret the sequences that i BLASTed?

Thank you.

#4 Trof

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Posted 07 March 2012 - 05:26 AM

Max ident percentage can be the same for several number of mismatches. The score (or the overal number of mismatches) is more important. If you have more sequences with the highest score, then take all of them.
Other option would be to search for published polymorphisms in those species, in your target sequence. But that may be difficult if you have many species, the database propably wouldn't be extensive for wide range of microorganisms.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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