One of the most important enzymes in the sequencing world, one which enables spectacular creativity on the part of novel assay designers, is Tn5 transposase. Personally, I spend many times each month thinking about how to use Tn5 and its ability to tagment - both tag and fragment - input DNA. There’s even reports that Tn5 can tagment RNA-DNA hybrids such as from reverse transcription or even long single-stranded DNA. I’ve covered seqWell in the past,with their fully kitted reagents; now the company (which just turned ten) is launching a Tagify product line that is focused on enabling NGS dreamers to easily explore new Tn5-based library preparation methods.
Tagmentation: A Versatile Reaction
Tagmentation lies at the heart of many NGS library prep workflows. Making short read library preparation simpler than ligation with Nextera was its original entree in to NGS, though Tn5-based Sanger protocols had existed in the past (though never particularly popular). Epicentreoriginally commercialized Nextera, then Illumina in a masterstroke bought Epicentre and immediately shut down sale of the 454-compatible kit. Oxford Nanopore would later introduce their rapid sequencing kit - so simple that even I can work it (badly, but I did get one usable read).
Since then, there have been a plethora of different applications. Look at what I’ve recently blogged - the Dovetail LinkPrep relies on tagmentation as does Scale Bio’s portfolio of library preps.. ATAC-Seq interrogates open chromatin and CUT&Tag explores transcription factor binding. There are even single cell and spatial versions of ATAC-Seq. TELL-Seq and single-tube Long Fragment Reads (stLFR) are very similar linked read technologies for getting long range information out of short reads. Several gene editing quality control schemes, such as UDiTaS, use tagmentation. There’s even some preps for Fiber-Seq for chromatin state interrogation methods that install PacBio SMRTbell adapters using tagmentation.
Tn5 transposase has many valuable properties. First thanks to the work of William Reznikoff and others, the biochemistry of the enzyme is well understood. Second, it will accept custom DNA payloads, so long as the specific Mosaic End sequences are present. Third, it is relatively tolerant to impurities in DNA - particularly important for crudely or not purified DNA such as within a droplet generated with 10X Chromium. Fourth, tagmentation is also efficient enough to require DNA inputs only in the low tens of nanograms or less - subpicogram can be achieved - if the library will be amplified. Fifth, as the portmanteau term tagmentation has been created for, it both tags and breaks DNA - leaving a nick that can either be repaired or often just played through - if the next step is PCR, then there is no melting portion in the first cycle!
If you want to play with this, you could express your own Tn5 transposase and load it. Be sure to use one of the high activity mutants - there are double and triple point mutants in the literature. Or there are suppliers that will sell you naked high activity Tn5 transposase - but you must load it with a correctly designed double-stranded DNA
Enter Tagify
Tagify from seqWell is offering ready-to-go Tn5, in three possible forms.
The non-custom option is high activity Tn5 carrying a P5 flowcell binding sequence plus a 10 basepair index (i5) plus a 10 basepair Unique Molecular Index (UMI) payload, available as a set of 24 or set of 96 such indices. This is useful whenever your wish to sequence whatever is next to something you know but you don’t know where it is. So think locations of random integrations of a known cassette, transposon insertion locations in Tn-Seq, or an adapter ligated to a double-strand break from on- or off-target CRISPR-directed cleavage (such in the UDiTaS assay). Or use your imagination. If you have a PCR primer targeting your known bit and also tagged with i7, then you can amplify and go straight onto a sequencer - with the UMI enabling accurate counting.
Option two is Tn5 transposase, but loaded with a custom payload of your design. So you have complete freedom to put in the sequence elements of interest and seqWell will ensure it is correctly loaded. The third option is like option two, but with seqWell’s engineered TnX enzyme. As seqWell CSO/founder Joe Mellor mentioned in his conversation with me, Tn5 transposase evolved for purposes which were not to be a great plaything for NGS enthusiasts. Protein engineering partner Codexis delivered an enzyme with higher specific activity - a larger fraction of loaded transposase perform their one-shot delivery to available DNA - and lower sequence specificity What new sequencing method will you develop with Tagify?
[2024-07-09 11:38 EDT corrected Joe Mellor title]
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