TITLE: Fast-Seq, a universal method for rapid and inexpensive genomic validation of rAAV vectors in preclinical settings
AUTHORS: Lucy Maynard, Olivia Smith, Nicolas Tilmans, Eleonore Tham, Shayan Hosseinzadeh, Ryan Leenay, Weilun Tan, Nicole Paulk

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sanger sequencing is the standard preclinical method used for sequence verification of AAV transfer vector cloning constructs and eventual packaged viral genomes. While Sanger sequencing does provide high quality data in a relatively short turnaround time, the data analysis requires manual evaluation of sequencing reads, rendering this approach incredibly low-throughput. Additionally, Sanger sequencing only provides 1-2X coverage, which results in low confidence single nucleotide polymorphism (SNP) and indel calling. It is imperative for preclinical gene therapy labs and core facilities to sequence both the transfer vector plasmids that go into viral production and the eventual packaged genomes to ensure no recombinations or mutations have occurred, and to validate that the genomes were not truncated during capsid packaging.</div><div class = "text-block">This protocol outlines a simple, fast, and inexpensive methodology geared toward preclinical environments for sequencing packaged AAV genomes via NGS, using paired-end Tn5 tagmentation-based library preparation. This protocol was optimized for use on the commonly available illumina MiSeq instrument, but other instruments would also work and should be chosen based on the number of samples and depth required (iSeq 100, MiniSeq, MiSeq, NextSeq, etc.).</div></div>

[STEPS]
?. [SOLUTIONS TO PREPARE IN ADVANCE OF AAV LIBRARY PREP AND STORE]
The dilution of each new batch of adapter-loaded Tn5 enzyme should be empirically determined based on activity. We recommend making a dilution series of loaded enzyme to determine the optimal yield and fragment size. Dilute the Tn5 enzyme in Tn5 Storage Buffer. For a starting point reference for your dilution series, we have found that adapter-loaded Tn5 at 1-ng/µL in storage buffer works best in our hands, however working concentration is subject to the activity of the batch. We suggest performing 2-fold serial dilutions in a range of concentrations both above and below this when qualifying your initial batch of Tn5. Adapter-loaded Tn5 lots can be custom ordered from the UC Berkeley QB3 MacroLab here: (http://qb3.berkeley.edu/macrolab/servicesrates/).(A) Preparation of 50-mL of Tn5 Storage BufferCombine all reagents for the Storage Buffer except for the glycerol, pH the buffer to 7.2, and then bring to a final volume of 20-mL. Sterile filter the buffer with a 0.22-µm filter. Then add 30-mL of sterile glycerol up to a final volume of 50-mL. Store at -20˚C.  ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED2NaCl5 M100 mM1 mL3EDTA0.5 M0.1 mM10 µL4HEPES-KOH, pH 7.21 M50 mM2.5 mL5DTT100 mM1 mM0.5 mL6Triton X-10098%0.1%51 µL7Glycerol100%60%30 mL8PCR-certified water--16 mL9Total volume--50 mLNOTE: To accurately pipette the Tn5 storage buffer to make dilutions of the Tn5 enzyme, heat the buffer in a 42˚C water bath, pipette the desired volume for the dilution, then cool the buffer on ice before making the dilution.(B) Preparation of 50-mL of 5X Tn5 Reaction BufferCombine the reagents for the Reaction Buffer, adjust the pH to 7.5, and bring to final volume of 50-mL. Sterile filter the buffer with a 0.22-µm filter. Store at -20˚C.  ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED2Tris, pH 7.01 M50 mM2.5 mL3MgCl2 1 M50 mM2.5 mL4PCR-certified water--45 mL5Total Volume--50 mL(C) Preparation of Neutralization BufferThe neutralization buffer is used to inactivate the Tn5 enzyme and quench the tagmentation reaction to stop over-tagmentation of the AAV libraries. Neutralization Buffer is a 0.1% Sodium Dodecyl Sulfate (SDS) solution in water. Heat the solution in a 37˚C water bath to completely dissolve the SDS and then cool to room temperature. Store at room temperature.(D) Qualify each new batch of Nextera adapter-loaded Tn5 enzymeThe dilution of each new batch of adapter-loaded Tn5 enzyme should be empirically determined based on activity. We recommend making a dilution series of loaded enzyme to determine the optimal yield and fragment size. Dilute the Tn5 enzyme in Tn5 Storage Buffer. For a starting point reference for your dilution series, we have found that adapter-loaded Tn5 at 1-ng/µL in storage buffer works best in our hands, however working concentration is subject to the activity of the batch. We suggest performing 2-fold serial dilutions in a range of concentrations both above and below this when qualifying your initial batch of Tn5. Adapter-loaded Tn5 lots can be custom ordered from the UC Berkeley QB3 MacroLab here: (http://qb3.berkeley.edu/macrolab/servicesrates/)
ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED2NaCl5 M100 mM1 mL3EDTA0.5 M0.1 mM10 µL4HEPES-KOH, pH 7.21 M50 mM2.5 mL5DTT100 mM1 mM0.5 mL6Triton X-10098%0.1%51 µL7Glycerol100%60%30 mL8PCR-certified water--16 mL9Total volume--50 mL
ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED2Tris, pH 7.01 M50 mM2.5 mL3MgCl2 1 M50 mM2.5 mL4PCR-certified water--45 mL5Total Volume--50 mL
?. [AAV TAGMENTATION WITH Tn5]
Background: The tagmentation reaction is a vital step for producing AAV libraries of uniform size. There are several buffer components necessary for this reaction. The Tn5 Storage Buffer is used to dilute the adapter-loaded Tn5 enzyme to the appropriate concentration. Tn5 enzyme is diluted to 1-ng/µL in Storage Buffer. Reaction Buffer is prepared as a 5X stock and is combined with a crowding agent, N,N-Dimethylformamide, to create the buffer that the Tn5 enzyme is added to for tagmenting.NOTE: The dilution of the adapter-loaded Tn5 should be empirically determined for every new batch of Tn5 enzyme based on activity. It is recommended to make a dilution series of pre-loaded Tn5 enzyme to determine optimal yield and fragment size.FOR 1 REACTION (multiply volumes to scale for more reactions):  2. Add 2.4-µL of Tagmentation Reaction Mix per PCR tube.  3. Add 1.6-µL of DNA solution per PCR tube.  4. Briefly spin down to pull all reagents to the bottom of the tube.  5. Tagment PCR tubes in a thermocycler with the following conditions: 1 cycle of 55˚C for 10 min; 1 cycle of 72˚C for 3 min; 1 cycle of 10˚C kept at an infinite hold.
?. [NEUTRALIZATION]
Add 0.8-µL of Neutralization Buffer per PCR tube.Spin down in a benchtop centrifuge/platefuge at room temp for 5-min at 4,000 rpm.
?. [INDEX PCR]
Add 2.4-µL of Index PCR Master Mix per PCR tube/well.Add 1.6-µL of premixed Illumina Nextera i5/i7 indexing primers previously diluted to 5-µM.Index 1 (i7);
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							CAAGCAGAAGACGGCATACGAGAT NNNNNNNNNNNN GTCTCGTGGGCTCGG
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							Index 2 (i5):
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							AATGATACGGCGACCACCGAGATCTACAC NNNNNNNNNNNN TCGTCGGCAGCGTC
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							bold = P5/P7 adapter sequence
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							italics = unique 12-bp barcode index
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							underline = primer for mosaic ends added during tagmentation
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							Additional help designing adapters can be found here:https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-adapter-sequences-1000000002694-09.pdf  3. Briefly spin down to pull reagents to the bottom of the tube.  4. Run the tubes in a thermocycler with the following conditions: 1 cycle of 72˚C for 3 min; 1 cycle of 95˚C for 30 sec; 12 cycles of (98˚C for 10 sec; 67˚C for 30 sec; 72˚C for 1 min); 1 cycle of 10˚C for an infinite hold.🛑 This is a safe stopping point 🛑
?. [QC LIBRARIES AND LOAD SEQUENCER]
1. QC pooled and cleaned libraries on a bioanalyzer for fragment size verification and sample integrity. Follow the manufacturer instructions for your bioanalyzer, and use their recommended "high sensitivity" DNA kit. QC pooled and cleaned libraries on a bioanalyzer for fragment size verification and sample integrity. Follow the manufacturer instructions for your bioanalyzer, and use their recommended "high sensitivity" DNA kit. The absolute shape and bp size of the peaks in the traces are less important than having a large smooth proportion of inserts in the 200 bp – 800 bp range. Inserts in this size range from AAV genomes are optimal for cluster formation on Illumina flow cells. 2. The fragment size range should be between 200 and 800 bp to effectively sequence the AAV genome. If this is not the case, review the AAV tagmentation step and determine if the generated library is over- or under-fragmented. Set up a qPCR to determine the library concentration. Although you can estimate the concentration with a Qubit, we highly recommend using qPCR instead as a gold standard method to determine the library concentration. We recommend following the detailed illumina protocol here:3. Load samples for sequencing according to the manufacturer's protocol from the instrument you're using. We have confirmed that samples prepared following this protocol run successfully on Illumina MiSeq using Nano V2 and iSeq instruments. For use on a MiSeq, prepare samples using a MiSeq Reagent Nano V2 kit, for 300 cycles, and run 2 x 75bp paired-end settings. Additional information on MiSeq specifications and expected outputs and run times can be found here: https://www.illumina.com/systems/sequencing-platforms/miseq/specifications.html
?. [SOLUTIONS TO PREPARE FRESH FOR AAV LIBRARY PREP]
NOTE BEFORE PREPARING SOLUTIONS:Before beginning: We've provided volumes for 1 sample. As these are small volumes susceptible to loss and evaporation, we recommend using PCR strip tubes rather than PCR plates, and making master mixes at least 1.5x the number of samples. For example, if making libraries of 25 samples, prepare master mixes that are suitable for 1.5 * 25 = 30 samples. This will ensure you don’t run out of master mix.(A) 1.6X Tris-DMF BufferIf this will be a regular assay, we recommend pre-mixing and aliquoting the 5X Tn5 Reaction Buffer and water and storing as aliquots. Importantly, DMF should be added fresh, right at the time of the assay.  AB1REAGENT1 SAMPLE (µL)25X Tn5 Reaction Buffer0.643PCR-certified water1.24100% DMF0.165Total Volume2(B) Tagmentation Reaction Mix  AB1REAGENT1 SAMPLE (µL)21.6X Tris-DMF Buffer23Tn5 Enzyme (adapter loaded)0.44Total volume2.4(C) Index PCR Master MixThis can be prepared while the AAV samples are tagmenting.  AB1REAGENT1 SAMPLE (µL)25X KAPA HiFi Fidelity Buffer1.63dNTP Mix0.244PCR-certified water0.45KAPA HiFi Polymerase0.166Total Volume2.4
AB1REAGENT1 SAMPLE (µL)25X Tn5 Reaction Buffer0.643PCR-certified water1.24100% DMF0.165Total Volume2
N,N-Dimethylformamide (DMF) is a volatile carcinogen and should be handled with appropriate safety precautions in a chemical fume hood. When handling DMF stock, wear appropriate PPE including a lab coat, goggles, closed toe shoes and double gloves. Dispose of any pipette tips that contact DMF in a labeled solid waste container. Clean pipettes with 70% ethanol after use with DMF.
AB1REAGENT1 SAMPLE (µL)21.6X Tris-DMF Buffer23Tn5 Enzyme (adapter loaded)0.44Total volume2.4
AB1REAGENT1 SAMPLE (µL)25X KAPA HiFi Fidelity Buffer1.63dNTP Mix0.244PCR-certified water0.45KAPA HiFi Polymerase0.166Total Volume2.4
?. [POOLING AND CLEANING AAV LIBRARIES]
1. Pool 8-µL of every well into a 1.5-mL DNA LoBind microcentrifuge tube. 2. To clean the pooled libraries of primer dimers and undesired fragment sizes, perform two SPRI-bead washes. a. Wash 1: 0.6X volume of your sample, followed with 2X wash with 80% ethanol, and elute into 100-µL EB. b. Wash 2: 0.7X volume of your sample, followed with 2X wash with 80% ethanol, and elute in 30-µL EB.3. Measure the final DNA concentration with a Qubit. Be sure to use Qubit thin-walled assay tubes and calibrate with the Qubit dsDNA High Sensitivity Assay Kit. NOTE: To have enough sample to run on an Illumina MiSeq, ensure that your final DNA concentration is at least 300-pM at this step to continue.4. If you're submitting samples to a core facility or external sequencing CRO for validation and sequencing, submit the samples at this stage (skip ahead to ‘DATA ANALYSIS’ once you’ve received the raw data back from your sequencing service provider). If you're planning on doing the validation and sequencing yourself, continue on to the next step.
🛑 This is a safe stopping point 🛑
?. [ISOLATING PURE ssDNA AAV GENOMES FROM VECTOR LOT]
STEPS1. We recommend extracting total gDNA from 1E11 full rAAV particles. However, this protocol has been successfully validated for inputs as low as 1E9 full rAAV particles. We suggest including a known pre-validated control vector with replicates for every run. To meet the minimum input DNA requirement for NGS, we recommend you prepare at least 8 samples (this can be composed of biological/technical replicates or dilutions of your sample and controls) for sequencing. 2. To remove unincorporated DNA, incubate rAAV samples with 40-U exonuclease and 10-U endonuclease for 30-min at 37°C in the provided 10X buffer, supplemented with 1 mmol/L of the provided ATP in a final volume of 200-µL in a protein low-binding microcentrifuge tube.  ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED210X Baseline ZERO buffer10X1X20 µL3Baseline ZERO endonuclease1U/uL10U10 µL4Plasmid-safe exonuclease10U/µL40U4 µL5ATP1mmol/L25mM8 µL6AAV 1E11 full viral particles-1E11x µL7PCR-certified waterx µL8Total Volume200 µL3. Incubate samples at 65°C for 10-min to stop the nuclease digestion.4. Continue with rAAV gDNA extraction with Qiagen's MinElute Virus Spin Kit following the manufacturer's protocol. Use the entire 200-µL reaction from Step 3 as the input for the kit. Additional reagents and equipment required for the kit: 100% ethanol, 1.5-mL microcentrifuge tubes, heat block, microcentrifuge, vortexer. Elute the final DNA into 28-µL, into a DNA low-binding tube and either store short-term (24-hr) at -20°C.🛑 This is a safe stopping point 🛑
Per NIH Biosafety Guidelines, recombinant AAV vectors are Biosafety Level 1 so long as the encoded transgene is not a potentially tumorigenic gene product or toxin molecule, and provided they were produced in the absence of a helper virus. Otherwise, it should be handled under Biosafety Level 2 containment.
ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED210X Baseline ZERO buffer10X1X20 µL3Baseline ZERO endonuclease1U/uL10U10 µL4Plasmid-safe exonuclease10U/µL40U4 µL5ATP1mmol/L25mM8 µL6AAV 1E11 full viral particles-1E11x µL7PCR-certified waterx µL8Total Volume200 µL
?. [SECOND STRAND DNA SYNTHESIS]
1. Heat the extracted gDNA input from above for 5-min at 95°C on a heat block then quench on ice. 2. Transfer the entire cold gDNA solution to a PCR tube and add a mix containing 6-µM of random primers, 2-mM of each dNTP and 10-U of DNA polymerase I (Klenow) in the provided buffer in a final volume of 50-µl.   ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED2Random primers60µM6µM5 µL3dNTPs10mM each2mM10 µL4Klenow5U/µL10U2 µL510X NEB2 buffer10X1X5 µL6gDNA from Step 1--28 µL7Total Volume50 µL3. Add sealed tubes to PCR to randomly prime DNA synthesis with a ramp of 0.1°C/second until 37°C, followed by 1 hour of incubation at 37°C. 4. If you are ready to go on to the next step, maintain your dsDNA AAV genomes on ice. If you won't be preparing your AAV libraries in the next 24 hours, store the tubes wrapped in parafilm at -20°C.🛑 This is a safe stopping point 🛑
ABCD1REAGENT[STOCK][FINAL]VOLUME NEEDED2Random primers60µM6µM5 µL3dNTPs10mM each2mM10 µL4Klenow5U/µL10U2 µL510X NEB2 buffer10X1X5 µL6gDNA from Step 1--28 µL7Total Volume50 µL
?. TROUBLESHOOTINGFinal DNA concentration is below 300-pM.Remeasure your DNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the input AAV gDNA concentration to 2-5 ng/µL, or increasing the number of PCR cycles. Another alternative is to increase the number of samples to be sequenced (we recommend a minimum of 8 samples/replicates). If your concentration is less than 1-ng/µL, even without diluting, increase the amount of input AAV. You can add more samples to your library by completing the protocol a second time with more samples (be sure to use unique indices), pooling the two libraries together and performing a final 0.7X SPRI clean-up, eluting into 30-µL. Fragment size as measured by the bioanalyzer is below 200 bp.Your DNA is over-fragmented. Possible causes are: a) you started with low-quality material. QC your starting material to ensure it is high quality DNA by absorbance; b) not enough starting material. Quantitate your starting DNA again by Qubit; c) your ratio of DNA to Tn5 is too low. Remeasure your dsDNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the dsDNA to 2-5ng/µL or increasing the number of samples to be sequenced. If the issue persists, we recommend requalifying your adapter-loaded Tn5 enzyme.Fragment size as measured by the bioanalyzer is above 800 bp.Your DNA is under-fragmented. Possible causes are: a) you started with too much DNA. Quantitate your starting DNA again by Qubit; b) your ratio of DNA to Tn5 is too high. Remeasure your dsDNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the tagmentation time at Step 13 from 10 minutes at 55 ̊C to 20 minutes, or decreasing the number of samples to be sequenced (a maximum of 50 AAV samples can be sequenced on a MiSeq chip); c) your Tn5 transposase enzyme is inhibited. Either requalify your adapter-loaded Tn5 enzyme or check your DNA purification kit (if you didn’t use the suggested Qiagen MinElute Virus Spin kit) for inhibitors.No library peak is present by bioanalyzer.This library should not be sequenced. Possible causes are: a) sample lost during bead clean up; b) low quality or insufficient starting DNA; c) not using a heated lid on the thermocycler during the second strand synthesis; or Tn5 transposase enzyme has expired and/or gone through multiple freeze thaws.How to qualify each new batch of Nextera adapter-loaded Tn5 enzyme.Remeasure your DNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the input AAV gDNA concentration to 2-5 ng/µL, or increasing the number of PCR cycles. Another alternative is to increase the number of samples to be sequenced (we recommend a minimum of 8 samples/replicates). If your concentration is less than 1-ng/µL, even without diluting, increase the amount of input AAV. You can add more samples to your library by completing the protocol a second time with more samples (be sure to use unique indices), pooling the two libraries together and performing a final 0.7X SPRI clean-up, eluting into 30-µL. 
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							Fragment size as measured by the bioanalyzer is below 200 bp.Your DNA is over-fragmented. Possible causes are: a) you started with low-quality material. QC your starting material to ensure it is high quality DNA by absorbance; b) not enough starting material. Quantitate your starting DNA again by Qubit; c) your ratio of DNA to Tn5 is too low. Remeasure your dsDNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the dsDNA to 2-5ng/µL or increasing the number of samples to be sequenced. If the issue persists, we recommend requalifying your adapter-loaded Tn5 enzyme.Fragment size as measured by the bioanalyzer is above 800 bp.Your DNA is under-fragmented. Possible causes are: a) you started with too much DNA. Quantitate your starting DNA again by Qubit; b) your ratio of DNA to Tn5 is too high. Remeasure your dsDNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the tagmentation time at Step 13 from 10 minutes at 55 ̊C to 20 minutes, or decreasing the number of samples to be sequenced (a maximum of 50 AAV samples can be sequenced on a MiSeq chip); c) your Tn5 transposase enzyme is inhibited. Either requalify your adapter-loaded Tn5 enzyme or check your DNA purification kit (if you didn’t use the suggested Qiagen MinElute Virus Spin kit) for inhibitors.No library peak is present by bioanalyzer.This library should not be sequenced. Possible causes are: a) sample lost during bead clean up; b) low quality or insufficient starting DNA; c) not using a heated lid on the thermocycler during the second strand synthesis; or Tn5 transposase enzyme has expired and/or gone through multiple freeze thaws.How to qualify each new batch of Nextera adapter-loaded Tn5 enzyme.Your DNA is over-fragmented. Possible causes are: a) you started with low-quality material. QC your starting material to ensure it is high quality DNA by absorbance; b) not enough starting material. Quantitate your starting DNA again by Qubit; c) your ratio of DNA to Tn5 is too low. Remeasure your dsDNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the dsDNA to 2-5ng/µL or increasing the number of samples to be sequenced. If the issue persists, we recommend requalifying your adapter-loaded Tn5 enzyme.
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							Fragment size as measured by the bioanalyzer is above 800 bp.Your DNA is under-fragmented. Possible causes are: a) you started with too much DNA. Quantitate your starting DNA again by Qubit; b) your ratio of DNA to Tn5 is too high. Remeasure your dsDNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the tagmentation time at Step 13 from 10 minutes at 55 ̊C to 20 minutes, or decreasing the number of samples to be sequenced (a maximum of 50 AAV samples can be sequenced on a MiSeq chip); c) your Tn5 transposase enzyme is inhibited. Either requalify your adapter-loaded Tn5 enzyme or check your DNA purification kit (if you didn’t use the suggested Qiagen MinElute Virus Spin kit) for inhibitors.No library peak is present by bioanalyzer.This library should not be sequenced. Possible causes are: a) sample lost during bead clean up; b) low quality or insufficient starting DNA; c) not using a heated lid on the thermocycler during the second strand synthesis; or Tn5 transposase enzyme has expired and/or gone through multiple freeze thaws.How to qualify each new batch of Nextera adapter-loaded Tn5 enzyme.Your DNA is under-fragmented. Possible causes are: a) you started with too much DNA. Quantitate your starting DNA again by Qubit; b) your ratio of DNA to Tn5 is too high. Remeasure your dsDNA concentration with a Qubit to ensure that concentration is indeed 1-ng/µL. If the concentration is 1-ng/µL, consider increasing the tagmentation time at Step 13 from 10 minutes at 55 ̊C to 20 minutes, or decreasing the number of samples to be sequenced (a maximum of 50 AAV samples can be sequenced on a MiSeq chip); c) your Tn5 transposase enzyme is inhibited. Either requalify your adapter-loaded Tn5 enzyme or check your DNA purification kit (if you didn’t use the suggested Qiagen MinElute Virus Spin kit) for inhibitors.
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							No library peak is present by bioanalyzer.This library should not be sequenced. Possible causes are: a) sample lost during bead clean up; b) low quality or insufficient starting DNA; c) not using a heated lid on the thermocycler during the second strand synthesis; or Tn5 transposase enzyme has expired and/or gone through multiple freeze thaws.How to qualify each new batch of Nextera adapter-loaded Tn5 enzyme.This library should not be sequenced. Possible causes are: a) sample lost during bead clean up; b) low quality or insufficient starting DNA; c) not using a heated lid on the thermocycler during the second strand synthesis; or Tn5 transposase enzyme has expired and/or gone through multiple freeze thaws.
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							Make a dilution series of Nextera adapter-loaded Tn5 enzyme in Tn5 Storage Buffer. We have found that 1-ng/µL is optimal for Fast-Seq. Use this concentration as middle starting position and test concentrations above and below this when qualifying your initial batch of Tn5. To then assess the efficacy of your various dilutions following fragmentation, perform a bioanalyzer run on all your dilution samples and see which concentrations of Tn5 give you fragments between 200 and 800 bp. Choose the lowest concentration that reproducibly works.
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							Additional help with troubleshooting common library preparation problems can be found here:
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							https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/nextera-xt-troubleshooting-technical-note.pdf
?. [QC LIBRARIES AND LOAD SEQUENCER]
NGS DATA ANALYSISWe have developed a free, open-source computational workflow for analysis of NGS data. Please go to the Paulk Lab Github repository to download our code and find instructions on how to run the pipeline in the README:https://github.com/paulk-lab/FastSeq-pipelineSummary: NGS fastq data files are filtered using Trimmomatic to remove adapter sequences, low quality reads (PHRED score ANTICIPATED OUTCOMES
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							This code outputs an Output folder in your data directory, containing sample-specific folders and a compiled .csv file containing NGS statistics for all samples. Each sample-specific folder contains all the files necessary for obtaining alignments and statistics. Of note, please take a look at the following files:
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							BAM alignment fileInferred consensus sequence in FASTA formatPDF depiction of your library prep fragment distributionRaw and filtered VCF file, containing all called SNPs and indels
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							Reported statistics include: mean coverage, standard deviation of coverage, number of SNPs/indels, mean fragment size, as well as other relevant metrics. Resultant BAM files can be aligned to your AAV transfer vector plasmid reference sequence and viewed in a genome browser, such as Integrative Genomics Viewer (IGV) which can be downloaded here:
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							https://software.broadinstitute.org/software/igv/downloadPlots of sequencing depth and coverage can be generated in R using ggplot, with instructions here: http://ggplot.yhathq.com/install.html
?. 1. Requirements:Computer with at least 2GB of RAM to be dedicated to Java heap, ideally 8GB+.DockerPython 3GitHub accountIf you haven’t already, get a GitHub account from here: https://github.com/joinDownload the Fast-Seq pipeline from the Paulk Lab Github: https://github.com/paulk-lab/FastSeq-pipeline*Critical step* Please read the GitHub README file before beginning for important instructions.To run the pipeline, simply type the following in the command line:python3 fastseq_pipeline.py  The data directory should contain all sequencing files, reference sequences and adapter sequences. The sequencing results should be in zipped FASTQ, the reference and adapters should be in FASTA. The adapters are trimmed using trimmomatic, so refer to documentation for trimmomatic for how to format the adapter file or mirror the included example.The CSV file should contain the following columns (order doesn't matter):SampleForward Read PathReverse Read PathAdapter PathReference PathEach of the paths should be relative to the data directory. For example, if the adapters are in data/adapters/adapter1.fa then the csv file should contain adapters/adapter1.faFinally, the script will generate a final stats file which contains a selection of statistics from picard and bcftools. This file will be output to the Output folder under the name final_stats.tsv.txtAn example is included. To run it, simply execute the following command in this directory:python3 fastseq_pipeline.py data example.csvSummary: Data will be returned in fastq format and filtered using Trimmomatic to remove adapter sequences, low quality reads (PHRED score ANTICIPATED OUTCOMESThis code outputs an Output folder in your data directory, containing sample-specific folders and a compiled .csv file containing NGS statistics for all samples. Each sample-specific folder contains all the files necessary for obtaining alignments and statistics.BAM alignment fileInferred consensus sequence in FASTA formatPDF depiction of your library prep fragment distributionRaw and filtered VCF file, containing all called SNPs and indelsIf you haven’t already, get a GitHub account from here: https://github.com/joinDownload the Fast-Seq pipeline from the Paulk Lab Github: https://github.com/paulk-lab/FastSeq-pipeline*Critical step* Please read the GitHub README file before beginning for important instructions.To run the pipeline, simply type the following in the command line:python3 fastseq_pipeline.py The data directory should contain all sequencing files, reference sequences and adapter sequences. The sequencing results should be in zipped FASTQ, the reference and adapters should be in FASTA. The adapters are trimmed using trimmomatic, so refer to documentation for trimmomatic for how to format the adapter file or mirror the included example.The CSV file should contain the following columns (order doesn't matter):SampleForward Read PathReverse Read PathAdapter PathReference PathEach of the paths should be relative to the data directory. For example, if the adapters are in data/adapters/adapter1.fa then the csv file should contain adapters/adapter1.faFinally, the script will generate a final stats file which contains a selection of statistics from picard and bcftools. This file will be output to the Output folder under the name final_stats.tsv.txtAn example is included. To run it, simply execute the following command in this directory:python3 fastseq_pipeline.py data example.csvSummary: Data will be returned in fastq format and filtered using Trimmomatic to remove adapter sequences, low quality reads (PHRED score ANTICIPATED OUTCOMESThis code outputs an Output folder in your data directory, containing sample-specific folders and a compiled .csv file containing NGS statistics for all samples. Each sample-specific folder contains all the files necessary for obtaining alignments and statistics.BAM alignment fileInferred consensus sequence in FASTA formatPDF depiction of your library prep fragment distributionRaw and filtered VCF file, containing all called SNPs and indelsEach of the paths should be relative to the data directory. For example, if the adapters are in data/adapters/adapter1.fa then the csv file should contain adapters/adapter1.faFinally, the script will generate a final stats file which contains a selection of statistics from picard and bcftools. This file will be output to the Output folder under the name final_stats.tsv.txtAn example is included. To run it, simply execute the following command in this directory:python3 fastseq_pipeline.py data example.csvSummary: Data will be returned in fastq format and filtered using Trimmomatic to remove adapter sequences, low quality reads (PHRED score ANTICIPATED OUTCOMES
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							This code outputs an Output folder in your data directory, containing sample-specific folders and a compiled .csv file containing NGS statistics for all samples. Each sample-specific folder contains all the files necessary for obtaining alignments and statistics.
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							BAM alignment fileInferred consensus sequence in FASTA formatPDF depiction of your library prep fragment distributionRaw and filtered VCF file, containing all called SNPs and indels
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							Reported statistics include: mean coverage, standard deviation of coverage, number of SNPs/indels, mean fragment size, as well as other relevant metrics. Resultant BAM files can be aligned to your AAV transfer vector plasmid reference sequence and viewed in a genome browser, such as Integrative Genomics Viewer (IGV) which can be downloaded here:
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							https://software.broadinstitute.org/software/igv/downloadPlots of sequencing depth and coverage can be generated in R using ggplot, with instructions here: http://ggplot.yhathq.com/install.html