Closing bacterial genome (7 contigs)

[Inma]

Hi all,

I am starting my PhD and am new in genome assembly. I started assembling 42 large contigs from 454 sequencing technology and I reduced the number to 7 but it is impossible to join the rest unless I use short-reads against the draft genome or something like this. Could anyone recomend any final step to close the genome? I am near...and a little bit desperated

Thank you in advance

Inma

[vineet jha]

you can try PCR, desing primer at end of contigs and do pcr. at max u will have to perform 14 PCR expt. measure the length of pcr product then you know , which segment is placed after which segment also you will know gap between the segment. replace the GAP by N and you will have your genome ready.

[boetsie]

It is highly possible that the contigs are seperated by (large) repeats. PCR is only applicable if they can cross over the repeats. Otherwise you will just know which contigs are connected by which repeat, but you do not know which normal contig comes after the repeat and thus you can not close the whole genome. Do you know what your contigs are, are some of them repeats (based on the number of reads aligning to them)?

Regards,
Boetsie

[Inma]

I tried PCRs when I had 13 contigs and reduced the number to 7 but any fragment is amplified performing PCRs in these 14 ends. I have used BWA to map the small reads to contigs and IGV to visualize them but it doesn't work to map reads between contigs and try to find a new assembly.

Regards,

Inma

[Inma]

Do you know how to detect assembly errors by mapping short reads against the contigs?

[SylvainL]

Hi Inma,

for microbial genome, I would warmly recommend Edena

http://www.genomic.ch/edena.php

And after, to fill-in the gaps, you can first check if it does concern ribosomal operons...

sylvain

[Inma]

Hi Sylvain,

Does Edena work with 454 reads?

Thank you,

Inma

[SylvainL]

Hi

sorry, it works with 128nt read max... Considering the higher rate of sequencing errors with 454 technology (compared to Illumina), you can first shorten your reads with fastx-trimmer function of fastx-toolkit.

Edena is really efficient, it may be worth trying it! The main critical point will be the number of reads...

Sylvain

[Inma]

I've had problems installing fastx-toolkit. Are there any other option to obtain "Illumina reads" from 454 ones?

Thanks in advance,

Inma

[SylvainL]

What is your system?

[Inma]

My system is Ubuntu 9.10

[SylvainL]

Ok, maybe it is hard time to upgrade to Ubuntu 12.04LTS...

Whatever, before upgrading, just go in the Ubuntu Software Center and type fastx-toolkit, it may allow an easy installation....

In case you do not succeed, just send me a private message, I will try to help you more tomorrow...

Cheers,

Sylvain

[Inma]

Hi all,

When 454 reads are trimmed, I lose much information, isn't it?

I did it and run Edena assembler but the new scaffolds are very differente to the first ones performed by Celera Assembler... :_(

Inma

[SylvainL]

Hi Inma,

of course you loose the part of the read you cut...

When you say you observe difference between the contigs obtained with Edena and the previously obtained with Celera, what do you mean? Are they different in sequence? One would expect they are shorter with Edena and then you may have more contigs as output with Edena...

[Inma]

Hi SylvainL

Yes, they are completely differents in sequence. Edena was run by taking only the 32 initials nucleotides from 454 reads.

Cheers,

Inma