[m.nyine]

Dear All,

Can the Newbler software help me to answer questions like How many SNPs and frequency of occurrence withing the cDNA sequences, INDELs, percent coverage for the already existing ESTs? I am generating a reference trancriptome but I have not reference genome sequence.

[edge]

Does anybody know how to evaluate the transcriptome assembly done by third party program is good or not?
Got any specify rules to follow?

Thanks.

[Marta]

How about BLASTX against RefSeq of related species or group? I always wanted to see uninterrupted ORFs of expected size. I also always check for the presence of the longest transcript expected in the particular species. The latest may not apply to all organisms

[edge]

Hi Marta,

Many thanks for your prompt reply.
I'm very appreciate it

Do you mind to explain a little bit more about your idea?
If I assembly my newly sequenced transcriptome sequence by using two different third party assembler (velvet, abyss, etc).
How can I know that which program able to assembly and get better transcriptome sequence?

Looking forward to your reply.
Thanks.

How about BLASTX against RefSeq of related species or group? I always wanted to see uninterrupted ORFs of expected size. I also always check for the presence of the longest transcript expected in the particular species. The latest may not apply to all organisms

[Marta]

Hi Edge,

you may want to try the following:

1. save the contigs created by each assembler in .fasta format. Or save just 1000 (or 100) longest contigs
2. download RefSeq proteins of your group of species to your machine
3. run BLASTX (you will need to install it on your machine, or run batch at the ncbi site) using your contigs as query and the downloaded RefSeq as database
4. parse the BLASTx output into a table so you can see the length of each query sequence (contig) and the length of the overlap from the BLAST output
5. I like to see that about 90% of my contigs are at about 3x of the overlap length

There are other QCs I do on assemblies, but this one would be the first filter.

I work for CLC bio, so I should point you to the product you can use to simplify such analysis. You can download the Genomics Workbench trial, that will allow you to run BLASTX and many other things from here:

http://www.clcbio.com/index.php?id=1292

Not sure if I can provide commercial links here when associated with the organization. I will hear from the site owner, I guess, if this is not OK :-)

[edge]

Hi Marta,

I'm getting the error message shown, "Fail to get main class"
Any idea or advice to solve this issue?
Thanks

[edge]

Hi Marta,

I got few more questions need your advice.
If the number of contigs that assembled by two different assembler are different, is it the best way is just select the top 1000 longest contigs?
Do you mind to explain a little bit more regarding "I like to see that about 90% of my contigs are at about 3x of the overlap length". I not sure regarding how to evaluate blastx result.

Currently I'm a research student, I know CLC bio, which do well in bioinformatic analysis

Many thanks for your reply.

[Marta]

Hi edge,

No sure what you are referring to in "Fail to get main class". You'll need to contact the support of the program you are using. If this is from CLC bio trial, write to [email protected]
Looks like it maybe Java related, but this is extend of my understanding.

Regarding the evaluation of assemblies. I have a very old, almost 3 years old presentation when we did comparison of assemblers and evaluated output produced by CLC de novo assembler. The presentation is very long, and available here:

https://docs.google.com/file/d/0B9g4lIAKxQTaMjgyN2UzYzgtNjViNy00MDIzLTkxOGYtZjI4YzQzMzQ5MzQz/edit

All you need is slide #25 and you will see what I mean

Best,

Marta