Long primers for illumina sequencing

[aka]

Hello, I was trying to run a pcr for downstream illumina sequencing; and the primers are specifically designed for this purpose (with Illumina adapters and barcodes), so the primers end up been very long (>65 bp).

The regular primers (without Illumina adapters and barcodes) worked pretty well. However, the products from Illumina primers look like smears but not clear bands on the gel. Anyone have encountered the same problem before? Any ideas about the optimization would help. Thanks!

[JamieHeather]

A simple way to get around any problems associated with long primers is to only use the long primers for as few cycles as possible, before doing the majority of your cycles using shorter primers at the very ends of your amplicons.

So, you can do say 1-4 cycles with your full-length primers (the fewer the better), purify, then amplify just with the outermost sequences. You can even do it all in the same reaction vessel, with the long primers in a limiting concentration so the shorter primers outcompete after their binding site has been added.

[microgirl123]

Illumina just published a bulletin with a 16s metagenomics protocol - https://my.illumina.com/MyIllumina/B...mic-sequencing. You can easily adapt it for any locus. We've been successfully using an adaptation of this for a while.

[TonyBrooks]

What are you attempting to amplify?
We successfully use our ~65bp primers to generate metagenomic libraries without problems (insert ~370bp).
The Illumina metagenomic method is way too expensive for us (twice as many PCR reactions plus Ilumina adapters are EXPENSIVE).

[Kumari Richa]

Hi,

I have used 515F/926R primers for amplifying hypervariable regions of 16SrRNA gene. I see multiple bands in the gel. Why is this so? Has anyone else experienced this? What is the other band(s) ?

Thanks
Richa

[pmiguel]

What are you attempting to amplify?
We successfully use our ~65bp primers to generate metagenomic libraries without problems (insert ~370bp).
The Illumina metagenomic method is way too expensive for us (twice as many PCR reactions plus Ilumina adapters are EXPENSIVE).

Hi Tony,
The step-out adapters are just oligos, so you could order them from some place other than Illumina.

Are the extra PCR reactions costly? It might work to add the internal, locus-specific, primers and the step-out (index + distal adapter) oligos straight into the initial reaction. Or would that just make a mess?

--
Phillip

[addyblanch]

Are you trying to do something like TraDIS? We had a go and ended up using abridged illumina primers for it to work successfully. But even with this when quantified using bioanyliser they were all different lengths. However it did produce data.

[TonyBrooks]

Hi Tony,
The step-out adapters are just oligos, so you could order them from some place other than Illumina.

Are the extra PCR reactions costly? It might work to add the internal, locus-specific, primers and the step-out (index + distal adapter) oligos straight into the initial reaction. Or would that just make a mess?

--
Phillip

We could have ordered the oligos separately and not used the Illumina ones.
PCR reactions aren't costly when you do a few - when you have 5,000 samples to process, then it becomes an issue and saving even small amounts per reaction add up to being able to do extra sequencing runs.
We did also thought about adding all four primers into one reaction but we were worried that funky things might start happening.
Our dual oligo/dual index approach is working well though, no problems with duff oligos or freeze-thaw issues yet and getting >80% >Q30 on a 500 cycle v2 kit.

[Mghanbari]

What are you attempting to amplify?
We successfully use our ~65bp primers to generate metagenomic libraries without problems (insert ~370bp).
The Illumina metagenomic method is way too expensive for us (twice as many PCR reactions plus Ilumina adapters are EXPENSIVE).

Hi Tony Brooks

I was wondering if you could let me know about the protocol you used for amplicon sequencing on MiSeq?

Thanks
Mahdi

[TonyBrooks]

I'll summarise below

The primers are as follows. I won't put the P5/P7 sequences in here as the are (c) Illumina. You should be able to find them by Googling. Make sure you use the paired end P5 & P7

P5[i5 Index]ACGTACGTACGTGGATTAGATACCCBRGTAGTC
P7[i7 Index]AGTCAGTCAGCCACGTCRTCCCCDCCTTCCTC

Once PCR is done, we clean up using 0.8X Ampure beads. We do two rounds of clean up, but because we're cheap, we reuse the beads. After you elute in 10mM Tris, add 0.8X of 2.5M NaCL/20% PEG-8000 to re-bind the DNA to the beads. Incubate for 5 mins, remove supernatent, wash 2X with 80% EtOH and finally elute in 30µL. We QC a random selection on the Bioanalyser DNA 1000 chip to make sure we have product and no dimer. Then we quantify with the Qubit and pool for sequencing.

The template specific part of the primers are in red. The sequences shown here are 16S 785F and 1175R for interrogation of V5-7. You can replace these with whatever you target, but you'll also need to redesign your read primers if you do.

The sequence upstream of this is random sequence that finds no match when we blast it. It allows us to also use long custom primers with the required Tm > 65ºC

Read 1 ACGTACGTACGTGGATTAGATACCCBRGTAGTC - spike 5µL of 100µM into well #12 of MiSeq cartridge
Index Read GAGGAAGGHGGGGAYGACGTGGCTGACTGACT - spike 5µL of 100µM primer into well #13 of MiSeq cartridge (rev comp of read 2)
Read 2 AGTCAGTCAGCCACGTCRTCCCCDCCTTCCTC - spike 5µL of 100µM into well #14 of MiSeq cartridge

We use extra long P10 tips in order to be sure we dispense the primers into the mix already in the cartridge. You have to spike in rather than use custom mixes so that the PhiX spike in (we use 10%) is also primed and read.

DON'T SET THE MISEQ UP TO USE CUSTOM PRIMERS!! This will make the MiSeq attempt to aspirate from wells #18-#20 which will be empty.

Then set the run up as per the MiSeq protocol.

[Mghanbari]

I'll summarise below

The primers are as follows. I won't put the P5/P7 sequences in here as the are (c) Illumina. You should be able to find them by Googling. Make sure you use the paired end P5 & P7

P5[i5 Index]ACGTACGTACGTGGATTAGATACCCBRGTAGTC
P7[i7 Index]AGTCAGTCAGCCACGTCRTCCCCDCCTTCCTC

Once PCR is done, we clean up using 0.8X Ampure beads. We do two rounds of clean up, but because we're cheap, we reuse the beads. After you elute in 10mM Tris, add 0.8X of 2.5M NaCL/20% PEG-8000 to re-bind the DNA to the beads. Incubate for 5 mins, remove supernatent, wash 2X with 80% EtOH and finally elute in 30µL. We QC a random selection on the Bioanalyser DNA 1000 chip to make sure we have product and no dimer. Then we quantify with the Qubit and pool for sequencing.

The template specific part of the primers are in red. The sequences shown here are 16S 785F and 1175R for interrogation of V5-7. You can replace these with whatever you target, but you'll also need to redesign your read primers if you do.

The sequence upstream of this is random sequence that finds no match when we blast it. It allows us to also use long custom primers with the required Tm > 65ºC

Read 1 ACGTACGTACGTGGATTAGATACCCBRGTAGTC - spike 5µL of 100µM into well #12 of MiSeq cartridge
Index Read GAGGAAGGHGGGGAYGACGTGGCTGACTGACT - spike 5µL of 100µM primer into well #13 of MiSeq cartridge (rev comp of read 2)
Read 2 AGTCAGTCAGCCACGTCRTCCCCDCCTTCCTC - spike 5µL of 100µM into well #14 of MiSeq cartridge

We use extra long P10 tips in order to be sure we dispense the primers into the mix already in the cartridge. You have to spike in rather than use custom mixes so that the PhiX spike in (we use 10%) is also primed and read.

DON'T SET THE MISEQ UP TO USE CUSTOM PRIMERS!! This will make the MiSeq attempt to aspirate from wells #18-#20 which will be empty.

Then set the run up as per the MiSeq protocol.

Many thanks Tony!