SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Illumina Adapter and Primer preparation S.Iyengar Sample Prep / Library Generation 8 05-30-2019 12:54 PM
Suggested approach for illumina MiSeq library preparation for microbial communities s Gian77 Illumina/Solexa 9 01-15-2014 01:46 PM
Miseq illumina primer rozitaa General 1 06-04-2013 04:02 AM
Miseq compatible in house library preparation method vl80 Illumina/Solexa 2 12-11-2012 05:15 AM
illumina library preparation and multiplexing jgibbons1 Illumina/Solexa 9 03-01-2010 01:29 AM

Reply
 
Thread Tools
Old 09-25-2014, 10:12 AM   #1
Cyrus Taheri
Junior Member
 
Location: Canada

Join Date: Aug 2014
Posts: 8
Question Illumina Miseq primer TM and library preparation for fungal metagenomics

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
Cyrus Taheri is offline   Reply With Quote
Old 09-26-2014, 02:35 AM   #2
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,248
Default

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.
nucacidhunter is offline   Reply With Quote
Old 09-29-2014, 02:34 PM   #3
Cyrus Taheri
Junior Member
 
Location: Canada

Join Date: Aug 2014
Posts: 8
Default

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.
Cyrus Taheri is offline   Reply With Quote
Old 09-29-2014, 04:59 PM   #4
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,248
Default

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.
nucacidhunter is offline   Reply With Quote
Old 10-01-2014, 09:03 AM   #5
Cyrus Taheri
Junior Member
 
Location: Canada

Join Date: Aug 2014
Posts: 8
Default

Thank you!
Cyrus Taheri is offline   Reply With Quote
Old 03-09-2015, 03:44 PM   #6
tjpott
Junior Member
 
Location: British Columbia

Join Date: Mar 2015
Posts: 2
Default

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.
tjpott is offline   Reply With Quote
Old 03-10-2015, 11:33 AM   #7
Cyrus Taheri
Junior Member
 
Location: Canada

Join Date: Aug 2014
Posts: 8
Default

Hi tjpott, no I have not tried Miseq on fungal ITS yet. What are your primers?
Cyrus Taheri is offline   Reply With Quote
Old 03-10-2015, 11:46 AM   #8
tjpott
Junior Member
 
Location: British Columbia

Join Date: Mar 2015
Posts: 2
Default

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.
tjpott is offline   Reply With Quote
Old 03-10-2015, 03:31 PM   #9
Cyrus Taheri
Junior Member
 
Location: Canada

Join Date: Aug 2014
Posts: 8
Default

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.
Cyrus Taheri is offline   Reply With Quote
Old 03-11-2015, 09:22 AM   #10
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,178
Default

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

Last edited by kmcarr; 03-11-2015 at 09:24 AM.
kmcarr is offline   Reply With Quote
Old 03-11-2015, 11:59 AM   #11
Cyrus Taheri
Junior Member
 
Location: Canada

Join Date: Aug 2014
Posts: 8
Default

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.
Cyrus Taheri is offline   Reply With Quote
Old 03-14-2015, 07:21 AM   #12
cement_head
Senior Member
 
Location: Oxford, Ohio

Join Date: Mar 2012
Posts: 256
Default

Quote:
Originally Posted by Cyrus Taheri View Post
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/art...l.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
Attached Files
File Type: pdf isme_j20128_combined.pdf (791.9 KB, 20 views)
cement_head is offline   Reply With Quote
Old 03-16-2015, 11:45 AM   #13
Cyrus Taheri
Junior Member
 
Location: Canada

Join Date: Aug 2014
Posts: 8
Default

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

Thank you,
Cyrus Taheri is offline   Reply With Quote
Reply

Tags
fungal primer, genomic dna concentration, illimina miseq, nextera xt tagmentation, primer melting temp.

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 10:10 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO