9mm Primer Size

[Jessica Wade]

Hi,

did anybody try to use a Primer of a total length of 85 nt for PCR?
up to now I only tried it with max. 75 nt total length without having problems.

Into a short DNA sequence I want to introduce several features so I'd need to run 2 separate sets of PCR following by two separately done ligation / transformation steps what is quite time consuming.

I have done upto 94-mer PCRs relatively easily. The template had been BAC, so I can not be sure if you are doing genomic PCR or something. In all long-primer PCRs, I have found that as long as the priming sequence is short (20-30 bp), and the rest is just adapters and enzyme sites, it works well.

Hallo boz from neighour lab use primer long 106 bp, it works but on the 3 end he has lost 13bp after amplification. If you overcame internal palindroms repeats, it should principialy work
good luck

Any length is fine really, it's just the likelihood of an error in the primer will be higher so don't be surprised if that happens. You can get them purified by HPLC which i think which separates primers by size (by the base pair) so that if any of them have an insertion or deletion that primer can be removed. Bit more expensive but it's an option if you're worried. Go for the one long primer, it should be fine.

as killerkoz17 said, any size if find. I have done PCR with 100bp primers many times before.

My lab skips the HPLC purification as we have never seen the use for it.

Primers are built 3' to 5' with a capping step between addition of each nucleotide. The capping reaction is very efficient, though the addition of a new nucleotide is less so (>98.5%) Consequently at the end of the synthesis run, as much as 60% of the primers made are truncated. Sounds bad, but as the primers are built 3' to 5', all these truncated primers are missing the 5'end. If a restriction site is placed on the 5' end of the primer (to ligate into a plasmid) and you can dispose of the HPLC purification.

Also it is important to avoid internal palindromic sequence and minimise primer self annealing. Of course this is not always possible but due try. And if there are small internal palindromes and self annealing, giving the primer a high annealing temperature helps.

The annealing temperature of a large primer is calculated by considering only the part of the primer that binds to the template. The annealing temperatures I use for large primer is 60 Celsius, (no less than 58 C and as high as 62 Celsius)

A useful website to check your primer is Oligo calculator by Northwestern, it calculate Tm, and spots areas which self anneal or form hairpin loops

Uniquely designed primers will lead to generation of specific amplicons. The length of the primers is usually 18-30 bases, because the random combination of this size of a primer will hit less than once per total genomic sequence (3 billion base pairs).04
For example,

A 17-mer or longer primer should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. Primers of this length generally are unique sequences in the human genome; however, it is important to ensure that portions of the primer do not have sequence or cross-homology with the target. Computer programs such as BLAST can be used to find regions of local similarity between sequences. The program compares nucleotide sequences to sequence databases and calculates the statistical significance of matches.

Primers longer than 30 bases do not demonstrate higher specificity. Additionally, long amplicons are more likely to cross-hybridize with other primers and sequences in the reaction mixture, and this can terminate the DNA polymerization.09

Hello everyone,
I have a questions regarding how to remove primer before running data2 (or use the trim command to remove primer). The primer set I used is 515f/806r The sequencing center told me that :

"the primers will vary a bit in size, that is, by 1, 2 or 3 bases. The reason is that Illumina's software does not do very good when the first few bases are the same, which is the case when we use the same universal primers for all the samples. So we (and others) have approached this by adding 1, 2, or 3 Ns at the beginning of the primer. That helps the software make the right base call and helps us obtaining more real data. "

So as a result, the primer F4 has three additional N compared to F1, F3 has two additional N compared to F1, and F2 has one additional N compared to F1. The final sequence length also varied from 291-294 bp. The sequencing center told me that they have already remove barcode. So what I need to remove is the primer (showing the letter after dash in the image).

I saw in the forum that people use --p-trim-left to remove primer. However, in my case, the position of my forward primer varied from 19 to 22 and the reverse primer varied from 20 to 23. If I using the following command : --p-trim-left-f-22 and --p-trim-left-r-23. They can remove all the primer but for some samples, additional Oligonucleotide can also be removed. If I did this, all the sequence after trim can also be different length since their original length is different and the length being remove is the same.

Do you think this is a good way (using Data2) to remove primer in my situations? Will this affect the other following analysis. Can you please give me some suggestions regarding how to remove the primers in my cases (either using Data 2 or other primer removal program).

You can absolutely use QIIME 2 to accomplish your goal of removing the primers and variable spacers. Just use cutadapt once; since the variable spacer is at the 5' end of the primer, just use the primer as the "front" adapter with cutadapt trim-single and the primers (and all preceding bases, including the variable spacer) will be removed. See the tutorial here and use the --help flag to see the full usage details for cutadapt:

So the commend --p-trim-left-f-22 and --p-trim-left-r-23 in Data 2 which can removed all the primers will not work in your opinion (since it will result in different length of the processed sequence)? I asked to one of my colleague and he told me that it is fine if you are OK to sacrifice some sequences. And Illumina sequencing itself cannot do good job at the beginning so the quality might be low and it is necessary to trim the the beginning part of the data. How do you think about this?

I also thought about the barcode method as you suggested. Do you think I can just consider those (extra spacer + primer) as barcode and create a text file to link them to specific samples and follow Demultiplexing steps in Qiime 2? My concern here is that then the barcodes will have three different length. Will the command --qiime demux can handle different length of barcodes.

I think what @Nicholas_Bokulich's solution suggests is that you don't worry about those spacers at all and simply put in your actual primer sequences which is the same in all your samples, since cutadapt will just remove everything (regardless of length) that preceeds that sequence.
For example if your primer is GTGCCAGCMGCCGCGGTAA (please properly double check this sequence from your files) and the spacers are N, NN, NNN before it, then cutadapt will look for GTGCCAGCMGCCGCGGTAA and remove that and everything else on the 5' of that, regardless of the length, so all of your Ns, (regardless of their true value) will be removed.

I have used the following cutadapt to trim in the (primer+extra spacer N) in my sequence. The primer I used is 515f/806r.
cutadapt trim-paired
--i-demultiplexed-sequences demux-paired-end.qza
--p-front-f GTGCCAGCMGCCGCGGTAA
--p-front-r GGACTACHVGGGTWTCTAAT
--verbose
--o-trimmed-sequences trimmed_remove_primers.qza
I compared the interactive quality plot of demux-paired-end.qzv (before remove the primers using cutadapt) with trim_remove_primers.qzv (after remove the primers using cutadapt). I am wondering why there are so much differences between the two plots. Did I do the remove primers steps correctly? More specifically, my questions are:

Question: The sequencing facility told me that "The products (primers included) should be around 291-294 bp" . But why I saw all blue box in the "demux-paired-end" plot (means equal length?) Why there are red box appeared after I used cutadapt? Did I remove primer correctly?

Questions: The quality plot of "demux-paired-end" looked strange compared to other quality plot I saw in the forum. @thermokarst mentioned in this thread that this kind of quality plot with narrow band of high-quality scores seems already be filter? Am I right? But I did not do any filtering steps. The "demux-paired-end" file is the file which I imported in Qiime 2 using commend qiime tools import. Since our sequencing facility told me that they already remove barcode so I did not go through the demultiplexing step.

Questions: If the red box make sense to appeared after removing primers. Do I need to get rid of all those red box by using --p-trunc-len when doing DADA2 filtering to make the sequence equal length (I am sorry if my questions is so basic to you. I am really trying hard to understood how to select the correct parameter for the DADA2 and understand the interactive quality plot. I have read many posts in the forum but still confused. )

No problem, patience is is the foundation of progress!
Your cutadapt command looks ok to me. We can tell because if you look into the length summary of those plots you will see that originally all your reads were about 251 nts long and after they are about 229-231 nts. That 20 nts difference is the length of your primers you trimmed. All good so far.

Those red boxes you speak of only appear in the tails of your quality plots and when you hover over them the description below changes so you can read what it is referring to. Those quality plots are produced by subsampling 10,000 reads out of all the reads you have. Should any of those reads not be as long as the rest/what the plot is showing you will see that warning informing you that some of your reads were not that long. In your forward reads this begins happening almost exactly at the same place where you would trim your adapters which makes sense. A very small percentage of your reads didn't have your primers in them so they were never trimmed. What those reads are is not exactly known, some sort of contamination or chimera perhaps but it shouldn't matter as those will likely be discarded downstream anyways. They also have much lower quality score as well so again I wouldn't worry about these since they'll be dealt with later. Unless something weird happens during your merging steps of DADA2 I'd say you're good.

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