Illumina Miseq primer TM and library preparation for fungal metagenomics

[Cyrus Taheri]

I have previously worked with 454, but fairly new to Miseq platform. Using Nextera XT sample preparation kit, I am going to study soil and root fungal communities using fungal specific primers with TMs of 56.4 °C and 52.7 °C (Illumina overhanger not included). Illumina 16 S protocol recommends using primers with TM of 60-65 °C. I have seen papers that have used primers with TMs less than 60 °C, but I am told using primers with lower TM may cause low sequencing yield and poor sequence quality. One solution would be to add few nucleotides to the primers, but I am concerned adding nucleotides may negatively affect amplification of some fungal taxa. I appreciate any thoughts/ suggestions in this regard.
In another matter, Illumina 16 S protocol suggests using an equal concentration of metagenomic DNA (5 ng/μl) for Amplicon PCR reactions. I am wondering if this is a strict requirement, I guess this concentration is too low for a successful PCR on environmental samples.
Thanks

[nucacidhunter]

To compensate for lower Tm of primers, annealing temperature for Amplicon PCR step (1st PCR) need to be appropriately adjusted. The rest of the protocol can be used as recommended. KAPA polymerase buffer has higher salt concentration and that should be taken into account when calculating primer Tm. The recommended input DNA is 0.5 ng/ul of PCR reaction and considering multiple copies of target genes that amount is sufficient. Using more input DNA can be detrimental to PCR if any inhibitors are present.

[Cyrus Taheri]

Hi nucacidhunter, thank you for your advise. I am more concerned if lower TM could affect the sequencing procedure in MiSeq. I agree that it is possible to adjust annealing temperature in first PCR, but I cannot think of any strategy to deal with issues raised from lower primer TM in SBS chemistry.

[nucacidhunter]

Low Tm would affect sequencing if you were using custom sequencing primers for sequencing in MiSeq. Illumina protocol uses standard Illumina sequencing primers and binding sites for them is incorporated into design.

[Cyrus Taheri]

Thank you!

[tjpott]

Hi Cyrus,

Were you at all successful with your sequencing run for fungal ITS? I am about to order primers for the same illumina protocol, but I'm also concerned about the melting temperature issues you brought up.

[Cyrus Taheri]

Hi tjpott, no I have not tried Miseq on fungal ITS yet. What are your primers?

[tjpott]

i'm using gITS7 and ITS4 (see Ihrmark et al 2012 New primers to amplify the fungal ITS2 region – evaluation by 454-sequencing). I'm trying to get fungal diversity estimates from decomposing root material.

I'm also multiplexing with 3 additional gene targets (cDNA). Apparently there is serious bias toward smaller read length (not surprisingly) on the miSeq platform. The product length for the above target averages ~330 bp, but ranges from 150-600 bp. PacBio is the only platform capable of resolving these long read issues. I'm currently exploring this option as the sequencing facilities here on campus have not been successful with fungal ITS.

[Cyrus Taheri]

You are right clustering tend to be biased toward smaller amplicons. Knowing that miseq platform has been around for a few years now, I am also a bit surprised why only few papers are published on fungal ITS using Miseq. Any clue why people at your campus did not get good fungal ITS sequences on Miseq? Do you know if they use the same primers as yours? I previously used 454 for ITS1 on wheat root fungal communities and it worked quite fine and since then they have improved read length a lot, so if miseq does not work for you, you might consider pyrosequencing.

[kmcarr]

We have good luck sequencing fugal ITS1 on the MiSeq using the primers described in

Philipp-André Schmidt, Miklós Bálint, Bastian Greshake, Cornelia Bandow, Jörg Römbke, Imke Schmitt. (2013) Illumina metabarcoding of a soil fungal community. Soil Biology and Biochemistry, 65:128-132, doi:10.1016/j.soilbio.2013.05.014

They generate a product ~200bp which includes all of ITS1.

[Cyrus Taheri]

Hi kmcarr, this is promising. May I ask you which chemistry are you using, 500 cycles v2 or 600 cycles v3 and how many cycles you run? Did you bioanalyze your final library, if yes what was the size range? Illumina 16S protocol suggests using 5% phiax, but I heard for low diversity libraries you need to add more, what phiax proportion you use? thanks.

[cement_head]

Hi kmcarr, this is promising. May I ask you which chemistry are you using, 500 cycles v2 or 600 cycles v3 and how many cycles you run? Did you bioanalyze your final library, if yes what was the size range? Illumina 16S protocol suggests using 5% phiax, but I heard for low diversity libraries you need to add more, what phiax proportion you use? thanks.

Hi,

For 16S and 18S emp based protocols we use 25% PhiX. We have not tried ITS regions yet but will do so soon using this reference:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090234

We use QIIME for analysis of 16S, 18S and other like amplicons. We use a Version 2 chemistry and the 300 rxn kit - sequencing 2x151bp + 12 bp index codes. The details of how to set all this up, including modifying the MiSeq config files, is in the Supplemental to the ISME paper (attached). Let me know if you have any questions.

Regards,
Andor

[Cyrus Taheri]

Thank you so much! I am eager to learn how your ITS sequencing trials go, good luck!

Thank you,