Newbler vector trimming issue

[andylai]

Hi all,

I put a suspected 3' primer into trimming database fasta file, looks like this
>trim_vector1
ACGAGCGGCCA

But when I running runAssembly with correct -vt path, it still indicate warning message:
Warning: Suspected 3' primer ACGAGCGGCCA, 52012 exact matches found.

Newbler would not display warning about others primers that I added in trimming database,
I am very confusing why Newbler only keep going give warning about this primer.

Could anyone give an explain? Whether it's a bug. I am using Newbler v2.3

[flxlex]

Strange. the adapters are still present in the contis?

Two things come to mind:
- you can only use the -vt option once, trying multiple -vt's will result in newbler using only one of them (can't remember if it was the first or last file when I did this)
- check the 454NewblerProgress file to see if newbler reports on having found your input file


Hope this helps!

[andylai]

Thanks for reply. I am not quite sure what do you mean -vt can only use one time. Now I got the out put result.
I tried to use:
cat 454AllContigs.fna | grep 'ACGAGCGGCCA'|wc -l

It get back result around 70. I think that means the adapter sequences still there in the result.

[andylai]

If I did not use -vt in the second time, runAssembly can not import the primerdatabase for removing primers.

[flxlex]

Thanks for reply. I am not quite sure what do you mean -vt can only use one time.

I mean something like this:

runAssembly -o name -vt primers1.fasta -vt primers2.fasta *.sff

In this case, either the first or second file will be used, but not both.

[zhengz]

Hi andylai,

Have you solved this problem? The problem seems to persist in v2.6.

[kopardev]

I encountered the same problem recently. The only way around was to trim the 3' adapter from the sff file using biopython and use the new trimmed sff file for assembly. This worked perfectly.

[vamosia]

You'll need to combined all the primer into one file. i.e

cat primers1.fasta primers2.fasta > primers.fasta

then used the combined primer file

runAssembly -o name -vt primers.fasta *.sff

my suspicious is newbler is only accepting on of -vt options but not both.