Sanger squencing

[nucacidhunter]

In some of my forward sequences there are only complementary motif. There is no expected motif. This is the real problem.

I am not sure if I correctly understand the issue. If you have cloned your PCR amplicons non-directionally into a vector then using vector primers for sequencing can result in sequencing forward or reverse strand. If you are sequencing amplicons directly using amplification primers you should get forward sequence with forward primer.

[niraj29]

In some of my forward sequences there are only complementary motif. There is no expected motif. This is the real problem.

[westerman]

If I understand your question, then yes, the motif and its complement could be in the forward strand. Doing a rev-comp of the forward strand would indeed bring back the motif and its complement.

[niraj29]

Thanks Rick !

Isn't it possible that the same sequence either forward or reverse could have the expected motif and its complement motif. For example AAATAAATAAAT_____________ATTTATTTATTTATTT (FORWARD sequence)

If this could be the case then it is also possible that there is no real expected motif in the forward sequence and when we reverse complement the forward sequence ATTT converts to AAAT. Isn't it?

[westerman]

Hello All,

I want to add a question.

I have forward and reverse sequence of amplified microsatellite region. Can we use the reverse complement option for forward sequence and assemble it with reverse sequence? I am asking this because I get the expected SSR motif by doing so. I will appreciate if anyone would answer this.

I don't see why not unless you are doing some sort of directional sequencing. Forward plus rev-comp of the reverse will equal the rev-comp of the forward plus reverse.

[niraj29]

Hello All,

I want to add a question.

I have forward and reverse sequence of amplified microsatellite region. Can we use the reverse complement option for forward sequence and assemble it with reverse sequence? I am asking this because I get the expected SSR motif by doing so. I will appreciate if anyone would answer this.

[sklages]

What's wrong with the other candidates mentioned?

[Desai]

Thank you for replying,

I have used both phred and cap3. This two are not perfect for my data and even not giving full contigs so is there any other tool which i can use?

[sklages]

Depending on the number of sequences, but for low input I'd go for Staden Package, http://staden.sourceforge.net/

Consed is a fine package (we used here for +10 years), but requires some experience in both setting up software in an Unix environment and the actual assembly itself.

my 2p.

[juan.isaza]

cap3
http://seq.cs.iastate.edu/
http://genome.cshlp.org/content/9/9/868.full

[westerman]

phred/phrap

http://www.phrap.org/phredphrapconsed.html

[Desai]

Dear Friends,

Hey I have just started to use data of saner seqencer(DNA analyser 370). I want to generate contig from forward and reverse sequence. I am very confuse that which assembler should I use. Is there anyone who has exaperience? I want free version tools only.

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Best Regard