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Old 01-17-2018, 02:15 PM   #1
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Location: USA

Join Date: Jan 2018
Posts: 1
Default trouble with nested-primer strategy --

Hi all,

I am a bit of a novice when it comes to bioinformatics and PCR, so I apologize in advance and will try to make my problem as clear as possible.

For our project, we extracted algal DNA from lake sediment cores. Our goal is to look at changes in algal communities through time. We were inspired by a study done in Qinghai Lake, China (Li et al 2016 Scientific Reports "Temporal Succession of Ancient Phytoplankton Community in Qinghai Lake and Implication for Paleo-environmental Change"). They used 23S rDNA (410 base pairs) in this study because it is present in both prokaryotic cyanobacteria and eukaryotic algae, and can be used to compare their relative abundances. That same year, another group published a study using a modified version of the same plastid 23S primers, and it resulted in a higher number of eukaryotic algal sequences by excluding proteobacterial sequences (Yoon et al 2016 PeerJ "Development of a cost-effective metabarcoding strategy for analysis of the marine phytoplankton community"). We decided to use this strategy in our analysis. The exact methods that we followed are quoted below from the Yoon et al 2016 study:

"To lower the PCR-based bias, a nested PCR strategy with low cycle numbers was adopted. Two forward primers (A23SrVF1:GGACARAAAGACCCTATG and A23SrVF2: CARAAAGACCCTATGMAGCT)and two reverse primers(A23SrVR1:AGATCAGCCTGT TATCC and A23SrVR2: TCAGCCTGTTATCCCTAG) were designed and synthesized (Macrogen, Republic of Korea). The first and second PCR amplifications were performed under the following cycling conditions: initial denaturation at 94 ◦C for 3 min, followed by 15 cycles at 94 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 30 s, with a final extension at 72 ◦C for 3 min. The first PCR reaction mixture (40 L) contained 10 ng of template, 2 L of each primer (20 pmol), 4 L of dNTPs (10 mM), 0.4 L Ex Taq Hot Start Version (2 U) (Takara Bio Inc., Shiga, Japan), and 4 L 10X buffer. The first amplicon was purified using the AccuPrep Gel Purification Kit (Bioneer, Republic of Korea) and eluted with 20 L elution buffer. The second PCR reaction mixture was the same as the first except that 2 L of purified PCR product from the first PCR amplification was used. As with the first round, 2 L of each primer was used for the second PCR (20 pmol). The products were separated by 1.5% agarose gel electrophoresis and stained with loading star (Dynebio, Sungnam, Republic of Korea). Amplicons of the expected sizes (approximately 410 bp) were purified using the Accuprep Gel Purification Kit (Bioneer, Daejeon, Republic of Korea). A library was constructed from the NGS data using the TruSeq Sample Preparation kit V2 (Illumina, USA). The quality and quantity of the library were measured using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and sequencing was performed using Illumina MiSeq (2 X 300 bp pair-ends) (Illumina, USA).”

So in summary, our PCR runs went as follows:
* 1st run using just the normal 23S primers.
* PCR cleanup and a 2nd PCR run using the inner primers from the manuscript with the fluidigm adaptors hooked on to each primer
* PCR cleanup again, quantify, dilute

We have been communicating with a laboratory that will be sequencing our samples since the summer. They told us: “If you wanted to do an experiment with 23S amplicons it would need to follow a similar approach; you would perform the primary PCR using CS1/CS2 target specific fusion primers and then ship those to us. We would add the indexed Illumina adapters, normalize, pool QC and sequence.”

We sent out samples out to that laboratory a few weeks ago, but they are now concerned about the product size of our amplicons. They say that when the fluidigm primers are used for barcoding, this will add approximately 120 bp to the products, resulting in products > 500bp. At this size, they will not be able to completely sequence the product with a 500 cycle run.

A longer explanation from them:

“When sequencing custom amplicons prepared using the two-step PCR method with Fluidigm CS1/CS2 oligomers on the MiSeq, primers complementary to CS1 & CS2 are added to the MiSeq reagent cartridge to server as primers for read 1 and read 2 respectively. The 5’ ends of each of these reads starts immediately at the junction between the CSx and your target specific sequences in the primers you used for the primary PCR. The span capable of being sequenced is between the 5’ positions of your target specific sequences. Using a MiSeq v2 500 cycle kit produces two (2) 250bp reads from those points towards the center of the fragment. To have sufficient overlap between these reads to merge them into one long pseudoread the recommendation is that the distance between the 5’ ends of the target specific portions of your primers is not > 400bp. Above 450bp is pushing your luck and if this distance is ≥ 500bp then no overlap is possible at all. “

When asked if the MiSeq v3 kit would be an option, I got this reply:

“Longer reads (2x300bp) are possible using the MiSeq v3 kit (apparently used in the method quoted). We normally will strongly recommend against using the long, v3 kits for amplicon sequencing. The quality of the 3’ ends of the reads generally becomes so degraded that you are will usually end up with only 2x250bp (or less) of usable data so don’t waste the money on the longer reads.”

At this point I am at a loss. I am not sure how we were able to get this far with the project only to learn that our amplicons are too long.

Is there a reasonable work-around that anyone can think of?
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