Question: Fungal genome sequencing
0
gravatar for salma.abdeljalil
4.4 years ago by
Tunisia
salma.abdeljalil0 wrote:

I am new to sequencing and I am wondering if it is  feasible to perform the sequencing using MiSeq machine with short libraries of 300 bp (paired-end sequencing) with Nextera kit..

I mean, can we do fungal whole genome sequencing using Illumina MiSeq and consequently, de novo assembly ? We want to perform  the sequencing of a fungal strain. The size of the genome is around 35 Mb...

Thank you :-)

miseq • 2.0k views
ADD COMMENTlink modified 4.4 years ago by lelle790 • written 4.4 years ago by salma.abdeljalil0
0
gravatar for thiagomafra
4.4 years ago by
thiagomafra70
Brazil
thiagomafra70 wrote:
Yes, it is perfectly possible make a fungal sequencing using MiSeq with Nextera kit. I used for yeasts (12mb) with great success! I recommend SOAPdenovo for de novo assembly.
ADD COMMENTlink modified 4.4 years ago • written 4.4 years ago by thiagomafra70

What k-mers you recommend?

ADD REPLYlink written 4.4 years ago by Adrian Pelin2.3k

It depends. There's a consensus which says that 60% of the size of the read is the optimal value of k-mer. Worked for me, but it is important to test different values. Start with 60%. Good luck.

ADD REPLYlink written 4.4 years ago by thiagomafra70
0
gravatar for lelle
4.4 years ago by
lelle790
Berlin
lelle790 wrote:

I have done an assembly of a fungi with a ~30mb genome from a Nextera MiSeq run and it did not go great. I ended up with a N50 of ~28,000. I think it should have worked better than that and I have not found any explanation why the assembly is not better.

I have talked to people that say the coverage of Nextera is not even enough for WGS. For example the Mira manual has a very strong opinion on the matter. I would appreciate comments about this.

ADD COMMENTlink written 4.4 years ago by lelle790

I did 250bp seq on 2 fungal genomes, same protocol, in one genome coverage was very uneven and bizzare, in the other genome the coverage was pretty even. The difference between the 2 genomes was size and GC content, the one with uneven coverage was larger 35 mb, and very low GC content, maybe ~20%.

ADD REPLYlink written 4.4 years ago by Adrian Pelin2.3k
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