I only jut now came across this thread, but can tell that the FuPa seems to consists of a blend of single stranded exonucleases and UDG/UNG (e.g. USER + ExoI see below).

The primers you get from ampliseq most likely contains urasils allowing the UDG/UNG to cleave the incorporated primer generating a 3'-overhang, which probably is removed by a 3' ssDNA exonuclease blunting the amplicon.

I have a very hard time accepting when the companies won't provide information about what is in their buffers/blends. I had an idea of what could be in the FuPa and tested it by buying some primers with and without urasils to allow generation of amplicons with urasils in the primer or not.

After FuPa the amplicons made with the urasil-primers had a length (checked by gel electrophoresis) corresponding to the amplicon without the primers while the amplicon made with the primers without urasils were full length.

You could probably make something similar to the FuPa by mixing the USER enzyme with exonuclease I although it might require some optimization ;-)

I did not try to ligate the adaptors, since this was just to satisfy my curiosity and find out what the FuPa contained.

Best regards,

Magnus

Hi there. The primers you are using are phosphorylated? If not you need to genereate 5`phosphorylated sites for ligation of your adapters. Of course this is only my educated guess because I have no experience with the Ion Torrent.

Cheers

The FuPa digestion is an elegant way to optimize the sequencing performance since you don't want to sequence 2 x 20 bp of your known sequencing primers for nothing, especially for any AmpliSeq approach. The FuPa reagent partially digests the primers and phosphorylates the amplicons. Although, I would love to know more about this (I assume) blend of exonucleases and what the acronym FuPa stands for.

I only jut now came across this thread, but can tell that the FuPa seems to consists of a blend of single stranded exonucleases and UDG/UNG (e.g. USER + ExoI see below).

The primers you get from ampliseq most likely contains urasils allowing the UDG/UNG to cleave the incorporated primer generating a 3'-overhang, which probably is removed by a 3' ssDNA exonuclease blunting the amplicon.

I have a very hard time accepting when the companies won't provide information about what is in their buffers/blends. I had an idea of what could be in the FuPa and tested it by buying some primers with and without urasils to allow generation of amplicons with urasils in the primer or not.

After FuPa the amplicons made with the urasil-primers had a length (checked by gel electrophoresis) corresponding to the amplicon without the primers while the amplicon made with the primers without urasils were full length.

You could probably make something similar to the FuPa by mixing the USER enzyme with exonuclease I although it might require some optimization ;-)

I did not try to ligate the adaptors, since this was just to satisfy my curiosity and find out what the FuPa contained.

Best regards,

Magnus

Primer digestion is essential to construct the library.....

FuPa:

My guess is

F = formamidopyrimidine DNA glycosylase (Fpg) to remove abasic site(s) created by cleaving uracil(s) in the primer)

u = USER enzyme to cleave the uracil

P = Polymerase (T4 DNA pol or Klenow) to fill in 5' overhangs and chew 3' overhangs

a = antibody to block Taq polymerase

US Patent US 8,728,728 B2 says on FuPa the following:

In some embodiments, phosphorylation of the amplicons can be conducted using a FuPa reagent. In some embodiments, the FuPa reagent can include a DNA polymerase, at least one uracil cleaving or modifying enzyme, an antibody and/or a storage buffer. In some embodiments, the FuPa reagent can further include at least one of the following: a preservative and/or a detergent. In some embodiments, the antibody is provided to inhibit the DNA polymerase and 3′-5′ exonuclease activities at ambient temperature.

Digest/Phosphorylate/Heat Kill the Amplicons

To the preamplified library (˜20 microliters), 2 microliters of FuPa reagent was added. Typically, the FuPa reagent comprises one or more uracil degradable enzymes such as UDG, FPG and the like, a DNA polymerase such as Pol I, T4PNK, Klenow and the like, and an antibody such as an anti-Taq antibody. In this example, the relative amount of each enzymatic component in the FuPa reagent was 1:1:1:1 but can be varied according to the number of required cycles, variations to the temperature profile, etc., as can be determined by one of ordinary skill in the art.

Digest/Phosphorylate/Heat Kill the Amplicons

To the preamplified library (˜20 microliters), 2 microliters of FuPa reagent was added. Typically, the FuPa reagent comprises one or more uracil degradable enzymes such as UDG, FPG and the like, a DNA polymerase such as Klenow fragment, and an antibody such as an anti-Taq antibody. In this example, the relative amount of each enzymatic component in the FuPa reagent was 1:1:1:2 but can be varied according to the number of required cycles, variations to the temperature profile, etc., as can be determined by one of ordinary skill in the art.

Example 21 Mutiplex Amplification with an Inosine Cleavable Group

In this example, target-specific primers containing one or more cleavable groups denoted as an inosine were prepared according to the primer selection criteria disclosed herein. After an initial in silico evaluation of the primer pairs to proposed target sequences, the evaluated target-specific primers (disclosed in Table 19—see U.S. Ser. No. 13/458,739, filed Apr. 27, 2012, hereby incorporated by reference in its entirety) were ordered from Integrated DNA Technologies (IDT)(Coravilla, Iowa). The inosine containing primers were received from IDT and subjected to multiplex amplification, performed according to the method of Example 15 with the following exceptions. The 2× Primer Pool (containing primer from Table 2—see U.S. Ser. No. 13/458,739, filed Apr. 27, 2012, hereby incorporated by reference in its entirety) was replaced with the Inosine containing primers (disclosed on Table 19 (see U.S. Ser. No. 13/458,739, filed Apr. 27, 2012, hereby incorporated by reference in its entirety), corresponding to SEQ ID NOs: 103122-103143). Additionally, after performing PCR amplification of genomic DNA targets, the samples was subjected to digestion with EndoV and FuPa.