TITLE: Tn5 Library Prep for Deep Sequencing Loci of CRISPR/Cas9 Edited Cells. Single gene specific primer amplification (UDiTaS protocol with alterations)
AUTHORS: Eric Danner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is the Tn5 gDNA prep published in the supplementary file of the UDiTaS paper (</span><a href="https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4561-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4561-9</span></a><span>).</span></div><div class = "text-block"><span>Usage: measuring chromosome fusions, InDels, mRNA splicing, structural variants, large resection, off target integration. -> Some of these DNA repair outcomes are demonstrated using such a sample preparation in this paper (</span><a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455975" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455975/</span></a><span> ). </span></div><div class = "text-block">My protocol is a modified version of UDiTaS but with a few tweaks like using nested primers. You will need a protein facility to produce the recombinant Tn5.</div><div class = "text-block"><span>Software for analysis is on </span><a href="https://github.com/ericdanner" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/ericdanner</span></a></div><div class = "text-block"><span>or </span><a href="https://github.com/editasmedicine/uditas" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/editasmedicine/u</span></a><span>ditas</span></div></div>

[STEPS]
?. [Reagent Preparation]
Order the correct DNA oligos   AB1WorkflowA.U.i5.N501UMITn5AATGATACGGCGACCACCGAGATCTACACTAGATCGCNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG2WorkflowA.U.i5.N(S)502UMITn5AATGATACGGCGACCACCGAGATCTACACCTCTCTATNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG3WorkflowA.U.i5_N(S)503UMITn5AATGATACGGCGACCACCGAGATCTACACTATCCTCTNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG4WorkflowB.U.i5.N501UMITn5AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG5WorkflowB.U.i5.N(S)502UMITn5AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNCTCTCTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG6WorkflowB.U.i5_N(S)503UMITn5AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTATCCTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG7U.Tn5-Abottom[Phos]CTGTCTCTTATACA[ddC]8U.P5.i5AATGATACGGCGACCACCGAGATCTACAC9Nested gene specific primer with overhang for i7 index addition:GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG‐[locus‐ specific sequence]10i7_N701CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATG11i7_N702CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGHere is an example amplicon with all of the correct ends ready for illumina sequencing.
AB1WorkflowA.U.i5.N501UMITn5AATGATACGGCGACCACCGAGATCTACACTAGATCGCNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG2WorkflowA.U.i5.N(S)502UMITn5AATGATACGGCGACCACCGAGATCTACACCTCTCTATNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG3WorkflowA.U.i5_N(S)503UMITn5AATGATACGGCGACCACCGAGATCTACACTATCCTCTNNNNNNNNNNTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG4WorkflowB.U.i5.N501UMITn5AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTAGATCGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG5WorkflowB.U.i5.N(S)502UMITn5AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNCTCTCTATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG6WorkflowB.U.i5_N(S)503UMITn5AATGATACGGCGACCACCGAGATCTACACNNNNNNNNNNTATCCTCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG7U.Tn5-Abottom[Phos]CTGTCTCTTATACA[ddC]8U.P5.i5AATGATACGGCGACCACCGAGATCTACAC9Nested gene specific primer with overhang for i7 index addition:GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG‐[locus‐ specific sequence]10i7_N701CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATG11i7_N702CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATG
?. [Reagent Preparation]
Make TAPS buffer5X TAPS-DMF: 50mM TAPS-NaOH @pH8.5, 25 mM MgCl2, 50% DMF-DMF- Sigma = D4551-250MLTAPS- Sigma = T5130-25G Mw- 243.2MgCl2 - mw 95.22DMF is unstable so make 10x and then dilute 1:1 on day of use. For 50ml (100mM TAPS, 50mM MgCl2 pH 8.5) 1.22g TAPS + 0.24g MgCl2. Raise to 40 ml. Titrate to pH8.5 w/ NaOH. Raise to 50ml. If it smells fishy discard.I have used it for 2 weeks without any issue after mixing.
?. [Reagent Preparation]
Adapter Oligo Annealing Re-suspend at 100uM in 10mM Tris-Hcl pH8, 0.5mM EDTA (edta inhibits Tn5 Mg2+ but it is so low in final working solution that it shouldn't matter). But might help  stabilize DNA that is baked for 12 hrs during annealing not degrade from nucleases. I have had issues with the DNA I resus in only Tris not working as well and I have no idea why- but maybe due to this.the final Mg2+ conc. is 5mM so even 0.5mM  EDTA shouldn't hurt it  massivelyTake 50ul of each primer (i5-UMI-TMI and Tn5A-bottom) and hybridize 12 hr. 95 C to cold ˜ 8min/1C
?. [Reagent Preparation]
Complex annealed oligos with Tn5 (Tn5 1.85mg/ml in 2x Tn5 dialysis buffer)100ul ul rxn: 85.7ul Tn5 + 14.3ul primer (original paper)116.6 ul rxn : 100ul Tn5 + 16.8ul primer (scaled)58.3 ul rxn: 50ul Tn5 + 8.4 ul primer23C on shaker (230rpm) for 60min. *shaking is key for  consistency * Mixing it by hand and leaving it on bench top produced variation of tagmentation quality. This variationtook me AGES to figure out.  Such a bad use of time. *Best to do larger 100ul rxn if possible. You need to do a titration exp to figure out reactivity and then you are left with a known working stock. 1 hr incubation at RT is what was used in the original Piscilli paper with this primer ratio. According to https://www.biorxiv.org/content/biorxiv/early/2019/03/06/568915.full.pdfthe Tn5-ProteinA fusion has the same hypermutated Tn5 protein, and is stable after primer complexing for 10 days at RT.
?. [Tagment DNA]
Clean up DNA before TagmentationImportant - I found Tn5 reaction inhibited by my gDNA extract. Not sure what was causing the inhibition but it goes away when you clean with SPRI beads.Remove RNA. The Tn5 can't react with RNA so probably don't need to remove it. But can easily add ul of 10mg/ml Rnase to 100ul DNA (i did 1ug/ul) in the TE buffer. Digest 10 min 37C shaker. Then the Nanodrop is more accurate. Wash with 1:1 SPRI beads to remove RNAse and any contamination. I washed 2x. Suspended in Tris-Cl and not EDTA. (or 0.1mM EDTA). EDTA inhibits the Tn5.
?. [Tagment DNA]
Measure gDNA  concentration.Use Qubit as the nanodrop is not accurate. RNA  contamination will make it seem like there is a lot more DNA than there actually is. However if you use Rnase, the Nanodrop can be accurate (check the 260/280 is 1.8).
?. [Tagment DNA]
Tagment gDNAYou can scale this and do a larger set if you have multiple genes to check on the same gDNA. Each tagmentation puts on its particular N50X index. But there are dozens of i7XX indexes so you can use a single i5 index, tagment it all together, and then use aliquots for the gene specific primer. Then for each sample give it a different i7 index.  AB1Reagents1 reaction2Nuclease free water4 µL35x TAPS-DMF4 µL4Genomic DNA (10 ng/µL)10 µL5Assembled transposome2 µL6Total reaction (µL)20 µL
AB1Reagents1 reaction2Nuclease free water4 µL35x TAPS-DMF4 µL4Genomic DNA (10 ng/µL)10 µL5Assembled transposome2 µL6Total reaction (µL)20 µL
?. [Tagment DNA]
Inhibit the Binding of Tn5 protein to tagmented DNAThere are 3 ways to proceed to remove the Tn5 connected to the DNA:1. Add 10ug (0.5ul of 20ug/ul) of Protinase K and incubate 10 minutes 55C. This is just for running on gel or bioanalzyer. Don't want protiinase K in the PCR reaction2. Fast but  I found  inconsistent: Use Znymo DNA conc columns. Don't use the MN columns as they require a volume too big for elution and also don't release the DNA as well. Add 100ul of DNA binding buffer. Then load on column. wash 2x with the wash buffer (200ul) and then  elute. No need to spin dry. Follow instructions in kit.3. Best: Use SPRI beads. 1:0.8 ratio (0,9X beads for 1 vol DNA).  Quench the 25ul reaction with 5ul of 0.2% SDS and put back on 55C for 7 min. Then increase vol to 100ul with water. Add 90ul beads (1: 0.9). Elute in 22ul Tris-Hcl 10mM (pH8).  You can scale up if you are going to do multiple PCRs off a prep
?. [Tagment DNA]
Test that tagmentation worked:1. Check by run on DS DNA High Sensitivity Bioanalzyer Chip.HS-DNA chip. It's hard to see this  accurately on a gel. You can see smears on a gel of 50-70ng tagmented gDNA (use reduced ladder amount if using gel as its very dim). You want 2ul of Tn5 to tagment 50ng of gDNA to about 2-3kb peak size.Bioanalyzer often fails. Make sure it has been recently cleaned. Scrub the electrode with DI water and a toothbrush. You want tagmented peak around 2-3kb.  and that while pressurizing the chip the syrninge should spring back to 0.6-0.7 2. Test your tagmentation functionally (w/ PCR). Take a tagmented 50ng gDNA SPRI cleaned and split it into 2 or 3 volumes and do PCR. i5 only to check background, i5 + primer1, and i5 + primer2. Then you can get an idea of the background tagmented gDNA.  It's important to do an only i5 control as if you over-tagment or if the i5 concentration is too high you get i5-i5 amplicons of the size that overlaps with your desired library size.Normally i5-i5 background amplicons are not made smaller than 1kb due to the suppresion PCR effect.
?. [PCR 1]
PCR1 amplify the target regionAmplify using gene specific primer. This is just a 20-25 nt primer to create amplicons.  This does not have the long 5' of the primer that binding of the i7 indexing primer. We will then do a nested PCR from these amplicons.The original Uditas protocol did not do a Nested PCR but any mis-priming event results in an amplicon as the i5 is universal.SPRI beads effect PCR so be sure to have no beads remaining in eluent. Even trace beads can effect PCR  ABC1Reagents1 reaction225 μl reaction50 μl reaction35X PrimeSTAR Buffer 5104TMAC (0.5M)1.53510 mM dNTPs (#N0447)246Gene Primer (10uM)0.517i5 Primer (10uM) prE3590.250.58OneTaq  DNA Polymerase0.519Template DNA102010Nuclease-free water5.2510.5Use a low concentration of i5 primer. You need to do control for i5 primer only to see where the  suppression PCR size limit is. Do this for every tagemented sampled. You do not want the i5-i5 amplicons to contaminated your specific PCRs.GXL Tm primer calculator:Tm (°C) = 2(NA + NT) + 4(NC + NG) - 5  (Ta (annealing) is  usually lower than Tm (melting))  When the Tm value (calculated by the following formula＊) is greater than 55℃, set the annealing temperature to 60℃. When the Tm value is 55℃ or less, set the annealing temperature to 55℃. ＊: Tm value calculation method: Tm (℃) = 2(NA + NT) + 4(NC + NG) - 5 where N represents the number of nucleotides in the primer having the specified identity (A, T, C, or G)the i5 primer is predicted to have a 69C annealing with GXL. So any temp should work from that side.  ABCDE1STEPTEMPTIME2Initial Denaturation98°C30 seconds312x       98°C                  60 °             C68°C10 seconds        15 seconds          60 sec4000x fold amp4Final Extension72°C3 minutes5Hold10C
ABC1Reagents1 reaction225 μl reaction50 μl reaction35X PrimeSTAR Buffer 5104TMAC (0.5M)1.53510 mM dNTPs (#N0447)246Gene Primer (10uM)0.517i5 Primer (10uM) prE3590.250.58OneTaq  DNA Polymerase0.519Template DNA102010Nuclease-free water5.2510.5
ABCDE1STEPTEMPTIME2Initial Denaturation98°C30 seconds312x       98°C                  60 °             C68°C10 seconds        15 seconds          60 sec4000x fold amp4Final Extension72°C3 minutes5Hold10C
?. [PCR 1]
Cleanup With Ampure XP SPRI beads 1. Use (1:1 vol). Can initially add 50 ul water to make the volumes bigger. 2. Elute in nuclease free water (25 ul) or TE.Can also use 0.5x  to get ride of >1kb bands before doing the normal clean up to remove the tagmented gDNA. The two figures shown below illustrate this idea.
?. [PCR 2 Nested]
PCR2.  Nested PCRThis is the nested PCR amplifying the amplicon you want to sequence. The gene specific primer contains a 5' sequence on it with a i7 adapter sequence.   ABCDE1Reagents1 reaction250 μl reaction35X PrimeSTAR Buffer 10410 mM dNTPs (#N0447)45TMAC (0.5M)36GSP primer (10uM) 17i5 Primer (10uM) prE3590.58OneTaq  DNA Polymerase19Round 1 product2510Nuclease-free water5.5  ABCDE1STEPTEMPTIME2Initial Denaturation98°C30seconds312x       98°C                       60C                        C68°C10 seconds          15 seconds              60 sec45Hold10C Clean up with Ampure 1:0.9 SPRIElute 25ul with 10mM Tris, 0.1 mM EDTA
ABCDE1Reagents1 reaction250 μl reaction35X PrimeSTAR Buffer 10410 mM dNTPs (#N0447)45TMAC (0.5M)36GSP primer (10uM) 17i5 Primer (10uM) prE3590.58OneTaq  DNA Polymerase19Round 1 product2510Nuclease-free water5.5
ABCDE1STEPTEMPTIME2Initial Denaturation98°C30seconds312x       98°C                       60C                        C68°C10 seconds          15 seconds              60 sec45Hold10C
?. [PCR- i7 indexing]
PCR-3: i7 indexingThis adds the i7 indexing sequence. The primers can be from the nextera kit or you can synthesize them yourself.   ABCD1Reagents1 reaction250 μl reaction35X PrimeSTAR Buffer 10410 mM dNTPs (#N0447)45i7 primer (10uM) Tm = 67 so use 60C for Ta2Uditas protocol stated crazy high conc but this is closer to what Illumina uses (Illumina is likely around 3ul of 10uM)6i5 Primer (10uM) prE35917OneTaq  DNA Polymerase18Round 1 product259Nuclease-free water7 For Nanodrop Nextera XT  Index Kit V2. The Nextera Kit says to use 5ul of their adapter for a 50ul reaction. Online I found someone saying the Nextera kits primers are about 5uM and you add 5 ul. So this is  The Overhang sequence between the gene specific primer and the i7 index is predicted 67C with GXL. The i5 primer is predicted 69C. So one can do the PCR as a 2step at 68C or 3 step at 65C.  ABCDE1STEPTEMPTIME2Initial Denaturation98°C30seconds310x - 12x       98°C                       65C                        C68°C10 seconds          15 seconds              60 secI have used 12 and don't see why not to use it as it maxes out nucleotides45Hold10C Clean up with Ampure 1:1 SPRI
ABCD1Reagents1 reaction250 μl reaction35X PrimeSTAR Buffer 10410 mM dNTPs (#N0447)45i7 primer (10uM) Tm = 67 so use 60C for Ta2Uditas protocol stated crazy high conc but this is closer to what Illumina uses (Illumina is likely around 3ul of 10uM)6i5 Primer (10uM) prE35917OneTaq  DNA Polymerase18Round 1 product259Nuclease-free water7
ABCDE1STEPTEMPTIME2Initial Denaturation98°C30seconds310x - 12x       98°C                       65C                        C68°C10 seconds          15 seconds              60 secI have used 12 and don't see why not to use it as it maxes out nucleotides45Hold10C
?. [PCR- i7 indexing]
Check with TOPO cloningIf this is a new protocol, TOPO clone some of your samples and align to finished sequences on the computer. Make sure all parts of your primers are there. Check where your sequencing primers will bind and make sure that the indexing primers read the i5 index before the i5 attached UMI's. Otherwise masking becomes complicated as your UMI is trapped between two indexing example amplicon with correct ends from my work.
?. [ Ilumina Sequencing]
Pool and Concentrate SamplesPooling and SPRI cleaned amplicons.1. Run your samples on a gel and cut out the size you want to sequence. Also run your i5 control on the gel to make sure your amplicons are not from background.The i5 added transposon adds 80bp. The overhangs/indexes off my gene specific primer for i7 are 66bp . So there is an extra of 146bp beyond the amplicon to be sequenced. If you want 200bp genomic read then sequence 346bp amplicon.2A. The most accurate way to measure concentration is qPCR using Kappa Quantification kit. This amplifies all amplicons that recieved the correct P5/P7 sequences. These are the sequences on the flow cell that will bind and amplify during sequencing.2B.  Also could calculate the molar amount of each product based on the concentration from Qubit and the size from the bioanalyzer.3.  Pool equimolar amounts of each product into a 1 mL Eppendorf tube.4.  Clean and concentrate pool using Agencourt Ampure XP SPRI beads (0.9x) Elute with 35 µL of 1x low TE
?. [ Ilumina Sequencing]
QPCR checkThis is the best way to quantify your amplicons and also show that they will functionally amplify in the sequencer flow cell. Kappa Quantification Kit . This will give a functional assay/quantification of your library. It uses the P5/P7 primers and you can then see how well your library amplifies, which will represent how it amplifies on the flow cell.I have found that my concentrations using size on Bioanalzyer/Qubit conc over estimates by 3-5x the functional concentration as measured by Qubit. And then when I sequence I see my samples are under represented compared to the PhiXCan also skip and just use bioanalyzer and QuBit but that is not recommended.
?. [ Ilumina Sequencing]
SequencingYou need to know the nM concentration of your sample. You will need to make 100ul of 1nM so you really don't need much.   10ng of 500bp is 32 fmol. So this would have to be diluted 32x to get the 1nM 10ng of 400bp is 40 fmol. We would have to dilute this 40x10ng of 300bp is 54 fmol. 1pmol/ul = 1uM. 1 fmol/ul = 1nMNeed to change the amount of indexing cycles from the normal 8 to 18.Read order: 1) Read 1 (from i5 index side do 141 bp read). 2) Index 1 - i7 index 8bp read. 3) index 2 read i5 side 18 bp. 8bp i5+ 10bp UMI. 4) read 2 primer goes from the i7 (do 151 bp). The gene specific primer was on the i7 side.
?. [ Ilumina Sequencing]
Check your runCheck your run BCL files with Sequence Analysis Viewer software from Illumina. Check the amount of aligned reads (your phiX reads). When you demultiplex you see a large 'undetermined' with indexes GGGGGGGG+AGATCTCG. This is phiX
?. [ Ilumina Sequencing]
Software for analysishttps://github.com/editasmedicine/uditashttps://github.com/ericdanner/uditas-replaceRequired files:for running uditas:Bowtie Index:One may compile your own bowtie2 index. But if your using only human or mouse then you can download the pre compiled and save a few hours.Download the .X.2bt files for your respective genome: http://bowtie-bio.sourceforge.net/bowtie2/index.shtmlhg38.1.bt2, hg38.2.bt2, etc (Make sure they are renamed to hg38.X.bt2 etc) Set enviornmental variable: bash command: export BOWTIE2_INDEXES=/file location2Bit genomes:Download hg38.2bit https://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips/hg38.2bitSet  environmental variable: bash command: export GENOMES_2BIT=/file location
?. [ Ilumina Sequencing]
BCL to Fastq conversionFor normal sequencing you can use the BCL2FASTQ software. But we added the UMIs in the indexing sequence so it alters the normal masking. Also the Uditas software has a pipeline for demultiplexing built in.If you want to just move the UMIs into the  beginning of read sequence 2 (not Uditas pipeline)change the readinfo.xmlor -use-bases-mask=y151,I8,I8,y151 command while running the BCL2FASTQ softwareBCL to unsorted FastQthis is what we use in our pipeline/ Uditas pipelinebcl2fastq \--runfolder-dir /input_directory--no-lane-splitting \--barcode-mismatches 1 \--use-bases-mask=Y151,I8,I18,Y141 \   #Y151,I8,I8,Y151 pushes UMI onto read2--create-fastq-for-index-reads \-p 8 \--output-dir /direcotry \--stats-dir /directory \--reports-dir /directory