some problems with the sequence analysis

Tolstova Olga Vl. Tolstova at mtu-net.ru
Sun Nov 17 23:05:33 EST 2002

Dear Colleagues!

I have some problems with making consensus.

My research director wants to have admirable quality consensus with the information about the heterozygote mutations, but hasn't the consensus quality criteria. I don't know them exactly either.

We are sequencing by ABI Prism 377 DNA Sequencer and use ABI Big Dye Terminator Cycle Sequencing Kit. The most part of PCR fragments has length about 400 bp. We have used Sequencing Analysis Software, Sequence Navigator, Vector NTI, ClustalX and MEGA programs for analysis our data.

My questions:

1) How I must make a consensus from the forward and reverse sequences? It goes without saying I rid of primers (if they have a place) and the unreadable ends. But what must I do with the parts, which are read only from the single sequence. Must I derive the consensus only from the part, which are read simultaneously from forward and reverse sequences or can I include in the consensus the parts, which are read well from single sequence too?

Which of my purpose can I fulfill with the consensus derived from the forward and reverse sequences simultaneously?

Which of my purpose can I fulfill with the compound consensus composed of the parts, which are read both only from the single sequence and from the forward and reverse sequences simultaneously (for subtyping, for the heterozygote mutation detection, for the publication)?

2) I am looking for the quantitative heterozygote criteria for PCR templates sequencing, but I haven't found out it yet.

What does it mean the sequence is good enough for the heterozygote mutation detection? (How many double peak percent has it? How long does it readable for detecting heterozygote mutation? ) Can I use the same program for detecting the heterozygote mutation?

As for Big Dye Terminator Cycle Sequencing Kit peak G is weak after peak A, and peak A is stronger after peak G. Vector NTI permits choose the "threshold for secondary base calling". How many percent does this level enough for admirable quality consensus? (I say here about 1.500 consensuses derived from HIV PCR templates).

 3) Can I translate my nucleotide consensus into amino acid sequence in any case (if I want to publish them)?

Do I need the first ATG-codon presence in my sequence?

How many frames must I use in order to translate into amino acid sequence, if I haven't the first ATG-codon in my sequence? Are they useful for anything (to publish the results of searching motifs, drug resistance sites or anything else)?

4) Can you advise me the literature?

Thank you for attention.

My e-mail is: Tolstova at mtu-net.ru

I look forward with pleasure to receiving your answer.

Yours faithfully,

 Tolstova Olga.

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