454 assembled reads

[katussa10]

I have 454 assembled contigs, Isotigs and singletons. The core facility that we used has done this for us, using Newbler. Do I still need to get rid of any adapter sequences or can I use these data directly for other analyses? In addition, how can I combine Isotigs, contigs and singltons?

[Khanjan]

The Singletons will still have the adaptor sequences. The contigs/isotigs mostly should not ( if at the time of running Newbler, a database of the adaptor sequences was specified ).

You can combine the singletons, contigs and the largest isotig from each isogroup and run it through one more assembly software ( Reference assembly if you have a reference genome ).

[katussa10]

Thanks Khanjan,
I don't have reference genome. In this case, is there a way to do the second assembly and what program do you recommend?
Thanks again.

[Khanjan]

You can use a genome which is closest to your organism. You can use Scarf as the Assembler.

[westerman]

The Singletons will still have the adaptor sequences.

I am not sure that follows. Singletons, per se, are not part of the Newbler output. The files 454Isotigs.fna and 454AllContigs.fna, sure, they exist. And the 454ReadStatus.tfa file tells where the reads went to. But there is no, as far as I know, Newbler generated singleton file.

We at Purdue Genomics do provide a Singleton.tfa file to our customers. The reads in this file have been trimmed using the data in 454TrimStatus.txt

[Khanjan]

I am not sure that follows. Singletons, per se, are not part of the Newbler output. The files 454Isotigs.fna and 454AllContigs.fna, sure, they exist. And the 454ReadStatus.tfa file tells where the reads went to. But there is no, as far as I know, Newbler generated singleton file.

We at Purdue Genomics do provide a Singleton.tfa file to our customers. The reads in this file have been trimmed using the data in 454TrimStatus.txt

Newbler does not generate the a file containing the singletons as it does for Isotigs and Contigs. But you can do a grep on the 454ReadStatus and find the read
ids of those which are singletons and then extract those singletons from the original sff files ( using sfffile/sffinfo )

Since the Singletons were not included in the Assembly, they wont be trimmed and will contain the adaptor.

[westerman]

Newbler does not generate the a file containing the singletons as it does for Isotigs and Contigs.

That was my point. You can not say where the singleton file came from.

Maybe the person who generated it just took reads marked 'singleton' from the 454ReadStatus file.

Or maybe the person who generated the file took the reads marked 'singleton' plus the information from the 454TrimStatus file in order to create a singleton file that is trimmed.

sfffile has the '-t' option (File containing accno/trim line information) so it is quite easy to do the trimming. We do this routinely.

But all in all you can not assume that the singleton is untrimmed. Nor trimmed.

[Khanjan]

That was my point. You can not say where the singleton file came from.

Maybe the person who generated it just took reads marked 'singleton' from the 454ReadStatus file.

Or maybe the person who generated the file took the reads marked 'singleton' plus the information from the 454TrimStatus file in order to create a singleton file that is trimmed.

sfffile has the '-t' option (File containing accno/trim line information) so it is quite easy to do the trimming. We do this routinely.

But all in all you can not assume that the singleton is untrimmed. Nor trimmed.

Yes, that makes sense. I assumed they are directly extracted from the *.sff file and not trimmed.

My sincere apologies