454 downstream analysis.

[aloliveira]

Hello,

I am working with 454 data and i have few question about the downstream analysis.

1) My data contains the adaptors sequences so i have to trimm these sequences out. How i proceed to do this?

2) I have reads with the adaptor and reads that did not have the adaptors sequences. How it's is possible i has reads with no adaptor if the adaptor itself who permits the sequencing reaction?

3) I discard the non-adaptors reads?

Best wishes,
André

[maubp]

Are you using the Roche "Newbler" tools? If so, you just provide you adapters or PCR primers in a FASTA file. And Roche already knows about their standard adapters and barcodes, so that is really easy.

[aloliveira]

Hy maubp,

I dont have the Newbler license. But i already know how to solve my problem. Anyway do you know the aswer for the second question? I dont know how its possible exists a read without adaptor sequence.

Thank you,
André

[maubp]

What kind of adapters?

What kind of downstream analysis (e.g. Mapping to a reference, de novo assembly, or reference guided assembly)?

[kmcarr]

Hello,

I am working with 454 data and i have few question about the downstream analysis.

1) My data contains the adaptors sequences so i have to trimm these sequences out. How i proceed to do this?

2) I have reads with the adaptor and reads that did not have the adaptors sequences. How it's is possible i has reads with no adaptor if the adaptor itself who permits the sequencing reaction?

3) I discard the non-adaptors reads?

Best wishes,
André

André,

Do you mean the 454 adapters themselves? You should never see these in your final reads. There are two primary reasons why you should not see them:

1) The sequencing primer starts at the keytag of the 3' end of the template (5' end of the output read) and then immediately proceeds into the inserted DNA. The software trims the keytag before outputting the final sequence so there is really no chance of having 454 adapter sequence at the 5' end of your read.

2) The library construction protocol should produce inserts which are longer than the average read length, it is unlikely that the read will reach the end of the insert and thus proceed into the 454 adapter at the 5' end of the template (3' end of the read). If by chance an insert is short and sequencing proceeds into the downstream adapter the 454 software will detect this and trim any adapter from the 3' end of your read.

Your final sequences thus should not contain the 454 adapters. If your library construction method used custom adapters then that is a different matter.

Hy maubp,

I dont have the Newbler license.
André

The Roche/454 Data Analysis software tools are available free upon request. You can request them from 454 at this website.

[Himalaya]

newbler quality trimming

Hi All
Does anyone has tried to do the quality trimming of 454 short sequence reads? My 454 short sequence reads are already trimmed for adaptors and MIDs. But there are poor quality bases that I wanted to trim. Anyone please help me out to do quality trim with Newbler...

Himalaya