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...
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.
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.
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.
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%.
What k-mers you recommend?
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.