Hi!

The 454 doesn't work nowadays, I mean, the company of 454 technology do not sell any more the reactive for use this machines. In the other hand, with Hiseq or Miseq Pair End you can obtain sequences of almost 550 bp. With Miseq you can have around 10 millions of sequences per run, and it could be more than enough for your research (for example, with the similar machine for 454 you only can obtain 100.000 sequences).

About the primers, if you want to focus on arbuscular mycorrhizal community, maybe it is better to use AMF primers, but with more universal primers you can have almost all the fungi.

Dear Dr. Jose Francisco,

Thank you very much for your answer and detailed explanation. 454 sounds like getting dispopular and not appropriate, so I will choose Illumina. Also, I was misunderstand about "pair end sequence" of Hiseq, 450bp flagment can be sequenced. I will choose appropriate AM specific primer pairs for this length.

I really appreciate for your big help.

PE sequencing is that they machine do the sequencing for both edges of the sequence (forward and reverse) and then you have to merge them to had the entire sequence. It allows you to have bigger sequences (~500-550 bp, if you do 300 PE),

Your illustration make my question clear.

Hiseq's PE sequencing length is 250 bp and my planning primer amplyfies 300 bp flagments of SSU.Therefore, I guess Hiseq can be applyed for this primer...

Dear Makoto, please take a look at this recent Nature paper: https://www.nature.com/articles/s41598-020-58942-3

Hi Makoto

This reference is useful for you, that mention about your work and in 2020, as follow:

"Perspectives of using Illumina MiSeq for identification of arbuscular mycorrhizal fungi"

Article Perspectives of using Illumina MiSeq for identification of a...

Also, this reference is useful

"The Influence of Bt Maize Cultivation on Communities of Arbuscular Mycorrhizal Fungi Revealed by MiSeq Sequencing"

https://www.frontiersin.org/articles/10.3389/fmicb.2018.03275/full