TITLE: Transposon insertion sequencing (Tn-seq) library preparation protocol - includes UMI for PCR duplicate removal
AUTHORS: Nagendra palani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Transposon insertion sequencing (Tn-Seq) is an NGS method to </span><span style = "font-style:italic;">en masse</span><span> map genomic locations of transposon insertions in a pool of mutants. Insertion mutants are usually made using synthetic mini transposons like Tn5 and Himar (in bacteria), Sleeping Beauty & Piggybac (in mammalian cells), and others like Mos (in Drosophila / </span><span style = "font-style:italic;">C. elegans</span><span>).</span></div><div class = "text-block"><span>There are several NGS methods to perform </span><span style = "font-style:italic;">de novo</span><span> Tn-Seq mapping of a library of mutants. Some of the key ones are listed below. </span></div><div class = "text-block"><span style = "font-weight:bold;">Van Opijnen, Tim, Kip L. Bodi, and Andrew Camilli. "Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms."</span><span style = "font-weight:bold;font-style:italic;">Nature methods </span><span style = "font-weight:bold;">6.10 (2009): 767.</span></div><div class = "text-block">    - Uses MmeI (a type IIs restriction enzyme) digestion to generate ~ 18bp genomic DNA overhangs from Mariner transposon insertions. Adapters are ligated to the fragments, the transposon - genome junction is enriched by PCR before being sequenced. Because of the short genomic sequence available for alignment to reference, mapping accuracy can be lower compared to fragmentation based library prep methods.</div><div class = "text-block"><span style = "font-weight:bold;">Langridge, Gemma C., et al. "Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants."</span><span style = "font-weight:bold;font-style:italic;">Genome research </span><span style = "font-weight:bold;">(2009).</span></div><div class = "text-block">    - DNA extracted from a pool of mutants is sheared/fragmented to a desired fragment size (usually 300 - 400 bp), fragments are end-repaired, and the transposon - genome junction is enriched by PCR before being sequenced.</div><div class = "text-block"><span>The list is only intended to highlight two major approaches to Tn-seq library prep - restriction digestion based or fragmentation based. There are several variations on each approach that are now available for the discerning scientist. There are also less known but interesting alternative methods like </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">HTML-PCR</span></a><span style = ":;">.</span></div><div class = "text-block">After trying out several of the above methods, we have developed a Tn-Seq library preparation protocol that incorporates desirable features from existing protocols and new features that will improve data quality, while being easy to perform (an undergraduate student with some wet-lab experience can go through the protocol in a day). </div><div class = "text-block"><span>The protocol is conceptually similar to the TraDIS protocol (see the Langridge </span><span style = "font-style:italic;">et al</span><span> paper above).</span></div><div class = "text-block">Key features are:</div><div class = "text-block">1. Uses the NEB Ultra II FS kit for enzymatic fragmentation of genomic DNA, end-repair of fragments, and ligation of adaptor. The kit allows performing all the reactions sequentially in a single tube (one-pot prep).</div><div class = "text-block">2. Incorporates Unique Molecular Identifiers (UMI) to enable removing PCR duplicates from sequencing data. Improves read count accuracy when the data is to be used for read-frequency based analyses (fitness or log fold change).</div><div class = "text-block">3. Includes 'best practices' library features like color diversity phasing and separate indexing step to save cost on adaptor oligos.</div><div class = "text-block">The method is also automation friendly.</div></div>

[STEPS]
?. [Adaptor formation]
Use 0.1x TE to prepare 100 μM stock of all the oligos.In a 0.2 ml PCR tube, add the following reagents, vortex well, and spin down. Place on the thermal cycler at  for   (heated lid).    -------------------------------------------- After incubation, terminate the incubation and let the heat block cool down to room temperature. Remove the tube and place it on the bench for 1 hr.The oligos will have annealed and formed the adaptor.In a 0.2 ml PCR tube, add the following reagents, vortex well, and spin down. Place on the thermal cycler at for  (heated lid). --------------------------------------------After incubation, terminate the incubation and let the heat block cool down to room temperature. Remove the tube and place it on the bench for 1 hr.The oligos will have annealed and formed the adaptor.
[Oligo TA_Adaptor_Top (100 uM)]
[Oligo  TA_Adaptor_Bottom (100 uM)]
[dH2O]
[Total]
?. [DNA Fragmentation, end-repair, ligation]
This section of the protocol is from the manual for NEB Ultra II FS kit - Catalog # E7805. (Refer to https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale7805.pdf : Section for Input > 100 ng for detailed protocol)Aliquot of genomic DNA into a well of a 96-well plate and use 1x TE to bring the volume to .Add the NEBNext Ultra II FS buffer + enzyme mix to the DNA (follow protocol from the linked manual)Set up the following program on the thermal cycler    for   for    hold. Add the NEBNext Ultra II ligation mix + enhancer to the well. Add  of the adaptor to the mixture. Mix well and incubate at   for  . Post incubation, add   of 0.1x TE and bring volume to  .Perform bead clean-up of the ligation mix using the sample purification beads that came with the kit. Set up the following program on the thermal cycler  for for  hold.Add the NEBNext Ultra II ligation mix + enhancer to the well. Add of the adaptor to the mixture. Mix well and incubate at  for . Post incubation, add  of 0.1x TE and bring volume to .Use  of beads for 1st bead addition of beads for 2nd bead addition (to select for an insert size distribution of 200 - 350 bp).Elute in of dH2O.
100 ng
26 µl
[heated lid]
This should fragment the library to have a distribution between 200 - 450 bp.
2 µl
[heated lid off]
28 µl
100 µl
30 µl
15 µl
10 µl
?. [Enrichment PCR]
Make 10 uM dilutions of the following primers.Transposon_Enrich_FwdTransposon_Enrich_RevFor each sample set up the following PCR in a 96-well plate.Transposon - genome junction enrichmentRun the following program on a thermal cycler.  - use heated lid10 - 15 cycles (to be decided by user)After end of PCR, use of the PCR end-product for Picogreen / Qubit.Useto run the sample on a Tapestation (D5000 high-sensitivity) to check for library size.Each sample library should be a unimodal distribution between ~ 300 bp - 600 bp. All libraries should have an average size distribution within 15% across all samples.
[Adaptor ligated DNA]
[Ultra II Q5 master mix]
[Transposon_Enrich_Fwd]
[Transposon_Enrich_Rev]
Use the NEB Tm Calculator to calculate the primer Tm values. If recommended Tm is greater than 65 ºC, use 65 ºC in the annealing & extension step below. For the Q5 master mix that comes with the NEB Ultra II FS kit (or the separately bought version), NEB recommends a combined annealing + extension step at 65 ºC. If your enrichment primers require a different annealing temperature, you can use the standard Q5 protocol (anneal at primer Tm, extend at 72 ºC). See here for more information).The number of PCR cycles needs to be decided by the user to get enough enriched DNA for indexing. Lower number of cycles is better to maintain sequence diversity.For the Q5 master mix that comes with the NEB Ultra II FS kit (or the separately bought version), NEB recommends a combined annealing + extension step at 65 ºC. If your enrichment primers require a different annealing temperature, you can use the standard Q5 protocol (anneal at primer Tm, extend at 72 ºC). See here for more information).
[Initial denaturation]
[Denaturation]
[Annealing & Extension]
[Hold]
1 µl
2 µl
?. [Indexing PCR]
Make 10 uM dilutions of the Nextera_R1 & Nextera_R2 primers.For each sample, use  of Enrichment PCR product as template for the indexing reaction.Set up the following PCR: As required---------------------------------------------------PCR Program:10 cyclesAfter end of PCR, use of the PCR end-product for Picogreen / Qubit.
10 ng
[Ultra II Q5 master mix]
[Nextera_R1]
[Nextera_R2]
[Template]
[dH2O]
[Total]
[Initial Denaturation]
[Denaturation]
[Annealing & Extension]
[Hold]
1 µl
?. [Library pooling, QC, and sequencing]
Pool samples by equal mass (assuming the library size distribution for the samples are similar). Otherwise, pool by equal molar mass.Do a 1.2x SPRI bead clean-up. Elute in  Check concentration of the pool by Picogreen/Qubit and quantify library using Kapa Illumina Library quantification kit. Dilute the pool to appropriate molarity required for the sequencing instrument.Load the pool on the sequencer and run with 15% PhiX spike-in for base diversity (works on MiSeq, HiSeq 2500, iSeq, and Novaseq. Might need more PhiX for NextSeq.)Do a 1.2x SPRI bead clean-up. Elute in Check concentration of the pool by Picogreen/Qubit and quantify library using Kapa Illumina Library quantification kit. Dilute the pool to appropriate molarity required for the sequencing instrument.Load the pool on the sequencer and run with 15% PhiX spike-in for base diversity (works on MiSeq, HiSeq 2500, iSeq, and Novaseq. Might need more PhiX for NextSeq.)Sequencing parameters : Paired-end sequencing with at least 100 base read length. Set Index read length appropriately.
[0.1x TE]
?. [Data analysis]
Scripts for pre-processing the data (fastq files to readcounts per insertion) will be made available at https://github.com/nppalani/TnSeq/ . The readcounts can then be consolidated and used with software like TRANSIT, TnSeqDiff etc., for gene essentiality analysis.Note that the scripts undergo frequent revision, so ensure that you are working with the latest version. Feel free to contact me if you have questions.