TITLE: ARTIC NEB Tagmentation protocol - high throughput whole genome sequencing of SARS-CoV-2
AUTHORS: Karan Bhatt, Amy Kistler, Angela Detweiler, Ashley Byrne, Gabrielle Lee, G Renuka Kumar, Lienna Chan, Lusajo Mwakibete, Michael B, Michelle Tan, Norma Neff, Sabrina Mann, Sharon Fong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for sequencing of SARS-CoV-2 in a 384 well plate format with miniaturized reaction volumes achieved with automated sample processing using liquid handlers. It has been used for all the SARS-CoV-2 genome sequencing associated with the Chan Zuckerberg Biohub COVID Tracker genomic epidemiology collaboration with California State and County Departments of Public Health from the period of May 2020 to June 2021. Extracted RNA is transcribed to cDNA, then amplified with ARTIC V3 primers to produce 400 bp amplicons that tile across the SARS-CoV-2 genome. Amplicons are tagmented using homebrew Tn5 transposase loaded with Nextera adapter oligos. The products of tagmentation are amplified with Nextera index primers to generate Illumina libraries for 2 x 150 bp (paired-end) sequencing on an Illumina sequencing platform.</div></div>

[STEPS]
?. [Input RNA prep - Dilution and plating]
High titer SARS-CoV-2 samples can exhaust-free ARTIC primers and form aberrant multimers during multiplex PCR (refer fig. 2). These larger amplicons can cause issues during downstream sequencing that impact genome sequence recovery. Hence, RNA samples need to be diluted by their Cycle Threshold or “Ct” values.(Note: RNA is temperature sensitive and prone to degradation. Follow RNAse-free practices and avoid multiple freeze-thaw cycles. Always place RNA plates on ice for intermediate steps and processes).We usually perform the following steps in a pre-PCR room. This space does not allow amplification of DNA. Follow the steps until the multiplex PCR mix and move the plate outside the pre-PCR room for cDNA amplification.Use liquid handlers like the Hamilton MICROLAB STARlet or similar to aliquot RNA samples from tubes or plates to a barcode-labeled 96 well plate (Biorad hard shell #HSP9601 or similar). Include a minimum of 1 negative control water aliquot on each plate. After generating a source plate of original RNA samples,  transfer an aliquot of each sample to a fresh plate for sequencing library prep. Use a Hamilton MICROLAB STARlet or similar liquid handler with a cherry-picking capability to transfer an aliquot of each RNA sample from the source plate into a fresh 'working' 96 well plate. As needed, dilute each individual RNA sample with DNAse/RNAse free water using the following schema:(Note: RNA is temperature sensitive and prone to degradation. Follow RNAse-free practices and avoid multiple freeze-thaw cycles. Always place RNA plates on ice for intermediate steps and processes).We usually perform the following steps in a pre-PCR room. This space does not allow amplification of DNA. Follow the steps until the multiplex PCR mix and move the plate outside the pre-PCR room for cDNA amplification.Use liquid handlers like the Hamilton MICROLAB STARlet or similar to aliquot RNA samples from tubes or plates to a barcode-labeled 96 well plate (Biorad hard shell #HSP9601 or similar). Include a minimum of 1 negative control water aliquot on each plate. After generating a source plate of original RNA samples,  transfer an aliquot of each sample to a fresh plate for sequencing library prep. Use a Hamilton MICROLAB STARlet or similar liquid handler with a cherry-picking capability to transfer an aliquot of each RNA sample from the source plate into a fresh 'working' 96 well plate. As needed, dilute each individual RNA sample with DNAse/RNAse free water using the following schema:Use liquid handlers like the Hamilton MICROLAB STARlet or similar to aliquot RNA samples from tubes or plates to a barcode-labeled 96 well plate (Biorad hard shell #HSP9601 or similar). Include a minimum of 1 negative control water aliquot on each plate. After generating a source plate of original RNA samples,  transfer an aliquot of each sample to a fresh plate for sequencing library prep. Use a Hamilton MICROLAB STARlet or similar liquid handler with a cherry-picking capability to transfer an aliquot of each RNA sample from the source plate into a fresh 'working' 96 well plate. As needed, dilute each individual RNA sample with DNAse/RNAse free water using the following schema:Cycle threshold valuesDilution1 - 15 1:1016 - 35 No dilution Label the final 96 well working plate with a minimum working volume of   of each diluted or undiluted sample as “W - barcode”.Use the Agilent Bravo or similar liquid handlers to transfer  of RNA from 4 x 96 well plates to a 384 well plate (Biorad - #HSP3901) as shown below. Affix a Library plate "L - 00x" barcode to this plate. In the event where you don’t have 384 samples, you can leave the wells empty or fill them with water as a control to trace contamination. (Note: for the Agilent Bravo,   of RNA is transferred to ensure a net of  is aliquoted to each well)♦ Checkpoint: you can potentially store this library prep plate at -80C. For optimal results continue until cDNA is made.  ♦
Cycle threshold valuesDilution1 - 15 1:1016 - 35 No dilution
14 µl
3 µl
3 µl
2.75 µl
?. [cDNA prep - First strand synthesis]
Before starting this step make sure the thermocycler is running the dNTP Hexamer priming program.Thaw reagents listed in table 2 on ice. Mix and spin down. Do not vortex enzymes.Make the master mix as described in table 2 inside a PCR hood. Alternatively, you can pre-make the master mix and aliquot in single-use Eppendorf tubes (store the pre-made master mix at -20ºC). Add   of master mix into wells A1-F1 of an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200). Seal the plate, spin down and let the reagents come to room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in the 384 well Library plate containing   of RNA.    Example protocol :  Carefully seal and centrifuge the plate briefly, then place the plate on the thermocycler. (Note: while the plate incubates in the thermocycler for 5 minutes make your master mix for the reverse transcriptase reaction.  Immediately cool the plate on ice for a minute and spin down.Thaw reagents listed in table 2 on ice. Mix and spin down. Do not vortex enzymes.Make the master mix as described in table 2 inside a PCR hood. Alternatively, you can pre-make the master mix and aliquot in single-use Eppendorf tubes (store the pre-made master mix at -20ºC). Add   of master mix into wells A1-F1 of an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200). Seal the plate, spin down and let the reagents come to room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in the 384 well Library plate containing   of RNA.    Example protocol :  Carefully seal and centrifuge the plate briefly, then place the plate on the thermocycler. (Note: while the plate incubates in the thermocycler for 5 minutes make your master mix for the reverse transcriptase reaction.  Immediately cool the plate on ice for a minute and spin down.Make the master mix as described in table 2 inside a PCR hood. Alternatively, you can pre-make the master mix and aliquot in single-use Eppendorf tubes (store the pre-made master mix at -20ºC). Add   of master mix into wells A1-F1 of an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200). Seal the plate, spin down and let the reagents come to room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in the 384 well Library plate containing   of RNA.    Example protocol :  Carefully seal and centrifuge the plate briefly, then place the plate on the thermocycler. (Note: while the plate incubates in the thermocycler for 5 minutes make your master mix for the reverse transcriptase reaction.  Immediately cool the plate on ice for a minute and spin down.Reagent 1x reaction volume (uL)400x reaction volume (uL)Notes 1.9 mM NEB Random Primers (E7422AA vial) 0.25170 70 uL added to account for dead volume 10 mM dNTP (N0447L)0.25170RNA Sample from step 12.75-Total3.25340Add  of master mix into wells A1-F1 of an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200).Seal the plate, spin down and let the reagents come to room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in the 384 well Library plate containing   of RNA.    Example protocol :  Carefully seal and centrifuge the plate briefly, then place the plate on the thermocycler. (Note: while the plate incubates in the thermocycler for 5 minutes make your master mix for the reverse transcriptase reaction.  Immediately cool the plate on ice for a minute and spin down.Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer  from the source plate to each well in the 384 well Library plate containing  of RNA.   # source wells:6wellsuL/well:54.6uLsource well transfer (nL)500nLExample protocol : Carefully seal and centrifuge the plate briefly, then place the plate on the thermocycler. (Note: while the plate incubates in the thermocycler for 5 minutes make your master mix for the reverse transcriptase reaction.  Immediately cool the plate on ice for a minute and spin down.(Note: while the plate incubates in the thermocycler for 5 minutes make your master mix for the reverse transcriptase reaction.  Immediately cool the plate on ice for a minute and spin down.Program dNTP Hexamer primingLid105°CVolume3.25 μLTempTimeStep 165°C 5 minStep 24°Chold
Reagent 1x reaction volume (uL)400x reaction volume (uL)Notes 1.9 mM NEB Random Primers (E7422AA vial) 0.25170 70 uL added to account for dead volume 10 mM dNTP (N0447L)0.25170RNA Sample from step 12.75-Total3.25340
54.6 µl
500
2.75 µl
# source wells:6wellsuL/well:54.6uLsource well transfer (nL)500nL
Program dNTP Hexamer primingLid105°CVolume3.25 μL
TempTimeStep 165°C 5 minStep 24°Chold
?. [cDNA prep - First strand synthesis]
Before starting this step make sure the thermocycler is running the reverse transcription program.On ice, thaw the reverse transcription reagents listed in the table 5. Mix and spin down. Do not vortex enzymes.Make up the master mix as described in table 5 inside a PCR hood.  Add   of master mix into each well for wells A3-P3 and A4-C4 of the same Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) used in step 2.  Seal the plate, spin down and let the reagents reach room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in a 384 well plate containing   of Annealed Template RNA.   Example protocol :  Carefully seal the plate, centrifuge briefly, and place the plate on the thermocycler. On ice, thaw the reverse transcription reagents listed in the table 5. Mix and spin down. Do not vortex enzymes.Make up the master mix as described in table 5 inside a PCR hood.  Add   of master mix into each well for wells A3-P3 and A4-C4 of the same Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) used in step 2.  Seal the plate, spin down and let the reagents reach room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in a 384 well plate containing   of Annealed Template RNA.   Example protocol :  Carefully seal the plate, centrifuge briefly, and place the plate on the thermocycler. Make up the master mix as described in table 5 inside a PCR hood.  Add   of master mix into each well for wells A3-P3 and A4-C4 of the same Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) used in step 2.  Seal the plate, spin down and let the reagents reach room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in a 384 well plate containing   of Annealed Template RNA.   Example protocol :  Carefully seal the plate, centrifuge briefly, and place the plate on the thermocycler. Reagent 1x Reaction Volume (uL)400x Reaction Volume (uL)Notes 5x SuperScript IV Buffer (Invitrogen - 18-090-200)1645Master mix is made for 645 reactions to account for dead volume.100mM DTT (Invitrogen - 18-090-200) 0.25161.25RNaseOUT RNase Inhibitor (Invitrogen - 10-777-019) (40 Units/ul)0.25161.25SuperScript IV Reverse Transcriptase (Invitrogen - #18-090-200) (200 Units/ul)0.25161.25Annealed Template RNA from step 23.25-Total51128.75Add  of master mix into each well for wells A3-P3 and A4-C4 of the same Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) used in step 2.Seal the plate, spin down and let the reagents reach room temperature before starting the Echo transfer. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer   from the source plate to each well in a 384 well plate containing   of Annealed Template RNA.   Example protocol :  Carefully seal the plate, centrifuge briefly, and place the plate on the thermocycler. Use the example protocol attached below as a reference to upload on the Labcyte Echo 550 machine. Using Labcyte Echo 550 or similar, transfer  from the source plate to each well in a 384 well plate containing  of Annealed Template RNA.  # source wells:19wellsuL/well:55uLsource well transfer1750nLExample protocol : ProgramReverse TranscriptionLid105°CVolume5μLTempTimeStep 125°C 5 minStep 242°C 50 minStep 370°C 10 minStep 44°Chold♦ Checkpoint: you can potentially store the cDNA plate at -20C. ♦
Reagent 1x Reaction Volume (uL)400x Reaction Volume (uL)Notes 5x SuperScript IV Buffer (Invitrogen - 18-090-200)1645Master mix is made for 645 reactions to account for dead volume.100mM DTT (Invitrogen - 18-090-200) 0.25161.25RNaseOUT RNase Inhibitor (Invitrogen - 10-777-019) (40 Units/ul)0.25161.25SuperScript IV Reverse Transcriptase (Invitrogen - #18-090-200) (200 Units/ul)0.25161.25Annealed Template RNA from step 23.25-Total51128.75
55 µl
1750
3.25 µl
# source wells:19wellsuL/well:55uLsource well transfer1750nL
ProgramReverse TranscriptionLid105°CVolume5μL
TempTimeStep 125°C 5 minStep 242°C 50 minStep 370°C 10 minStep 44°Chold
?. [Multiplex - cDNA amplification]
The ARTIC primer list (V3) can be found here - https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.tsv. These can be purchased in a pool or as individual primers. If purchased as individual primers, resuspend as described in the attached file below:Before starting this step, make sure the thermocycler is running the Multiplex PCR program.♦ Thermocycler should be outside the pre-PCR room ♦Prepare multiplex PCR master mix for pool 1 and pool 2 using the table below :  Using a SPT Labtech Dragonfly or similar liquid handler, dispense   of the Pool 1 and Pool 2 PCR master mix into a 384 well plate labeled L-00X-POOL-1 and L-00X-POOL-2  respectively.Make sure to use different syringes and reservoirs for the Dragonfly runs. Place the L-00X-POOL-1 and the L-00X-POOL-2 plates on ice.Using the Agilent Bravo or similar liquid handler, transfer  of the cDNA from step 3 to the L-00X-POOL-1 and L-00X-POOL-2 plates respectively. Seal plates, spin down and take plates to a thermocycler in the post-PCR lab space. ♦ Thermocycler should be outside the pre-PCR room ♦Prepare multiplex PCR master mix for pool 1 and pool 2 using the table below :  Using a SPT Labtech Dragonfly or similar liquid handler, dispense   of the Pool 1 and Pool 2 PCR master mix into a 384 well plate labeled L-00X-POOL-1 and L-00X-POOL-2  respectively.Make sure to use different syringes and reservoirs for the Dragonfly runs. Place the L-00X-POOL-1 and the L-00X-POOL-2 plates on ice.Using the Agilent Bravo or similar liquid handler, transfer  of the cDNA from step 3 to the L-00X-POOL-1 and L-00X-POOL-2 plates respectively. Seal plates, spin down and take plates to a thermocycler in the post-PCR lab space. POOL 11x reaction volume (uL)400x reaction volume (uL)NotesNEBNext Ultra II Q5 Hot Start 2x Master Mix (M0544L)2.51115.00master mix is made for 446 reactions to account for dead volumePrimer Pool 1 (10 μM)0.8368.00Nuclease-free Water0.292.00cDNA from step 31.5-Total 51610POOL 21x reaction volume (uL)400x reaction volume (ul)

NotesNEBNext Ultra II Q5 Hot Start 2x Master Mix (M0544L)2.51115.00master mix is made for 446 reactions to account for dead volumePrimer Pool 2 (10 μM)0.8368.00Nuclease-free Water0.292.00cDNA from step 31.5-Total 51610Using a SPT Labtech Dragonfly or similar liquid handler, dispense  of the Pool 1 and Pool 2 PCR master mix into a 384 well plate labeled L-00X-POOL-1 and L-00X-POOL-2  respectively.Make sure to use different syringes and reservoirs for the Dragonfly runs. Place the L-00X-POOL-1 and the L-00X-POOL-2 plates on ice.Using the Agilent Bravo or similar liquid handler, transfer  of the cDNA from step 3 to the L-00X-POOL-1 and L-00X-POOL-2 plates respectively. Seal plates, spin down and take plates to a thermocycler in the post-PCR lab space. Using the Agilent Bravo or similar liquid handler, transfer of the cDNA from step 3 to the L-00X-POOL-1 and L-00X-POOL-2 plates respectively. ProgramMultiplex PCRLid105°CVolume5μLStepsTempTimeCycleStep 198°C 30 sec1Step 298°C15 sec30*Step 363°C5 minStep 44°Chold1♦ Checkpoint: you can potentially store the multiplex PCR plate at -20C overnight. ♦.
POOL 11x reaction volume (uL)400x reaction volume (uL)NotesNEBNext Ultra II Q5 Hot Start 2x Master Mix (M0544L)2.51115.00master mix is made for 446 reactions to account for dead volumePrimer Pool 1 (10 μM)0.8368.00Nuclease-free Water0.292.00cDNA from step 31.5-Total 51610
POOL 21x reaction volume (uL)400x reaction volume (ul)

NotesNEBNext Ultra II Q5 Hot Start 2x Master Mix (M0544L)2.51115.00master mix is made for 446 reactions to account for dead volumePrimer Pool 2 (10 μM)0.8368.00Nuclease-free Water0.292.00cDNA from step 31.5-Total 51610
3.5 µl
1.5 µl
ProgramMultiplex PCRLid105°CVolume5μL
StepsTempTimeCycleStep 198°C 30 sec1Step 298°C15 sec30*Step 363°C5 minStep 44°Chold1
?. [Multiplex - Pooling and Dilution of PCR products]
♦ Pooling:At this stage, amplicons are highly concentrated and can easily aerosolize causing cross-contamination. Before proceeding, centrifuge the multiplex plates (L-00X-POOL-1 and L-00X-POOL-2) for a few minutes. This will minimize potential contamination.    Using an Integra or similar liquid handler, transfer   from source L-00X-POOL-1 plate to a fresh destination plate labeled as L-00X-POOL-1+2.Repeat the same   transfer from L-00X-POOL-2 source plate to the same L-00X-POOL-1+2 destination plate. Do not mix using the Integra. Carefully seal the L-00X-POOL-1+2 destination plate, and spin down at high speed for 2 minutes. In the end, you should have   to work with in the L-00X-POOL-1+2 plate. ♦ 1:100 Dilution:Note: For the 1:100 dilution of pooled multiplex PCR reactions in L-00X-POOL-1+2 plate, we use an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200), because it can hold more volume than the 384 Biorad HS PCR plate and allows us to perform a 1:100 dilution in a single step. Alternatively, you can do serial dilutions to achieve 1:100 dilution with Biorad HS PCR plates. Using an Integra or similar liquid handler, transfer    of nuclease-free water to an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) destination plate. This can be pre-made and stored at 4°C. Using an Integra or similar liquid handler, transfer    from the L-00X-POOL-1+2 plate to the pre-made Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) with   of nuclease-free water in it. Label the plate as L-00X-POOL-1+2-1:100. Carefully seal and spin down the L-00X-POOL-1+2-1:100 plate.Using an Integra or similar liquid handler, transfer  from source L-00X-POOL-1 plate to a fresh destination plate labeled as L-00X-POOL-1+2.Repeat the same  transfer from L-00X-POOL-2 source plate to the same L-00X-POOL-1+2 destination plate. Do not mix using the Integra. Carefully seal the L-00X-POOL-1+2 destination plate, and spin down at high speed for 2 minutes. In the end, you should have  to work with in the L-00X-POOL-1+2 plate. ♦ 1:100 Dilution:Note: For the 1:100 dilution of pooled multiplex PCR reactions in L-00X-POOL-1+2 plate, we use an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200), because it can hold more volume than the 384 Biorad HS PCR plate and allows us to perform a 1:100 dilution in a single step. Alternatively, you can do serial dilutions to achieve 1:100 dilution with Biorad HS PCR plates. Using an Integra or similar liquid handler, transfer   of nuclease-free water to an Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) destination plate. This can be pre-made and stored at 4°C. Using an Integra or similar liquid handler, transfer   from the L-00X-POOL-1+2 plate to the pre-made Echo Qualified 384-Well Polypropylene 2.0 Plus Microplate (# PPL-0200) with  of nuclease-free water in it. Label the plate as L-00X-POOL-1+2-1:100. Transfer  of the diluted pool from the L-00X-POOL-1+2-1:100 plate to a new Biorad HS 384 well PCR plate using an Integra or similar liquid handler. This transfer accounts for dead volume. The actual volume transferred is .
2 µl
2 µl
4 µl
49.5 µl
0.5 µl
49.5 µl
0.5 µl
0.4 µl
?. [Tagmentation - Fragmentation and Adaptor ligation]
CZBiohub uses a homebrew recombinant Tn5 transposase to fragment the DNA amplicons and ligate adapter sequences compatible with the Nextera dual index sequencing primers for Illumina sequencing. This protocol is modified from previously reported tagmentation-based protocols. [1,2,3]Pre-make a 5X stock solution of tagmentation buffer ("TAG"), and store at -20°C. Dilute to a final working concentration of 2.18X from 5X and aliquot in single-use Eppendorf tubes and store at -20°C. Make 1X Tn5 master mix using the following recipe:   1x Reaction Volume (uL)Master Mix (uL)Amplicon DNA0.4TAG Buffer (From 2.18X stock)0.9142857143512.00DMF0.0844.80HB Tn50.2057142857115.20Total1.6672.00More information to test homebrew Tn5 batch:     # source wells:16uL/well:38.4Pipette into wells A1-P1 of a new 384-well plate  Before making the master mix, make sure the thermocycler is running the Tagmentation program.    In a chemical fume hood, mix DMF and TAG Buffer. Add homebrew recombinant Tn5 to the buffer mix. Move quickly to make the master mix.NOTES:  Tn5 is temperature-sensitive and can precipitate out of solution. Handle with care and avoid multiple freeze-thaw cycles. Stocks of homebrew recombinant Tn5 are diluted in a 50% glycerol mix. It is critical to slowly pipette up and down with a wide-bore or P1000 pipette to thoroughly mix the solution. DO NOT VORTEX. On ice, manually pipette    of the Tn5 master Mix into wells A1-P1 of a Biorad HS 384-well PCR plate. Seal and spin down briefly. With the SPT Labtech Mosquito or similar liquid handler, use the Tn5 reaction mix plate as the source plate and the L-00X-POOL-1+2-1:100 plate from step 5 as the destination plate, transfer   of Tn5 reaction mix to each well of the destination plate.Immediately seal the destination plate, spin down and place it on the thermocycler. Save the source plate to re-use in step 7.  ProgramTagmentationLid105°CVolume2μL     TempTimeStep 155°C 5minStep 24°Chold While the Tagmentation program is running, start making the source plate for the tagmentation neutralization mixture (you can use the same plate as the Tn5 source plate with different columns).Pre-make a 5X stock solution of tagmentation buffer ("TAG"), and store at -20°C. Dilute to a final working concentration of 2.18X from 5X and aliquot in single-use Eppendorf tubes and store at -20°C. Make 1X Tn5 master mix using the following recipe:   1x Reaction Volume (uL)Master Mix (uL)Amplicon DNA0.4TAG Buffer (From 2.18X stock)0.9142857143512.00DMF0.0844.80HB Tn50.2057142857115.20Total1.6672.00More information to test homebrew Tn5 batch:     # source wells:16uL/well:38.4Pipette into wells A1-P1 of a new 384-well plate  Before making the master mix, make sure the thermocycler is running the Tagmentation program.    In a chemical fume hood, mix DMF and TAG Buffer. Add homebrew recombinant Tn5 to the buffer mix. Move quickly to make the master mix.NOTES:  Tn5 is temperature-sensitive and can precipitate out of solution. Handle with care and avoid multiple freeze-thaw cycles. Stocks of homebrew recombinant Tn5 are diluted in a 50% glycerol mix. It is critical to slowly pipette up and down with a wide-bore or P1000 pipette to thoroughly mix the solution. DO NOT VORTEX. On ice, manually pipette    of the Tn5 master Mix into wells A1-P1 of a Biorad HS 384-well PCR plate. Seal and spin down briefly. With the SPT Labtech Mosquito or similar liquid handler, use the Tn5 reaction mix plate as the source plate and the L-00X-POOL-1+2-1:100 plate from step 5 as the destination plate, transfer   of Tn5 reaction mix to each well of the destination plate.Immediately seal the destination plate, spin down and place it on the thermocycler. Save the source plate to re-use in step 7.  ProgramTagmentationLid105°CVolume2μL     TempTimeStep 155°C 5minStep 24°Chold While the Tagmentation program is running, start making the source plate for the tagmentation neutralization mixture (you can use the same plate as the Tn5 source plate with different columns).Tris-HCl, pH 7.4 (Lonza, 51247)50 mMMgCl2 (Invitrogen, AM9530G)50 mMMake 1X Tn5 master mix using the following recipe:   1x Reaction Volume (uL)Master Mix (uL)Amplicon DNA0.4TAG Buffer (From 2.18X stock)0.9142857143512.00DMF0.0844.80HB Tn50.2057142857115.20Total1.6672.00More information to test homebrew Tn5 batch:     # source wells:16uL/well:38.4Pipette into wells A1-P1 of a new 384-well plate  Before making the master mix, make sure the thermocycler is running the Tagmentation program.    In a chemical fume hood, mix DMF and TAG Buffer. Add homebrew recombinant Tn5 to the buffer mix. Move quickly to make the master mix.NOTES:  Tn5 is temperature-sensitive and can precipitate out of solution. Handle with care and avoid multiple freeze-thaw cycles. Stocks of homebrew recombinant Tn5 are diluted in a 50% glycerol mix. It is critical to slowly pipette up and down with a wide-bore or P1000 pipette to thoroughly mix the solution. DO NOT VORTEX. On ice, manually pipette    of the Tn5 master Mix into wells A1-P1 of a Biorad HS 384-well PCR plate. Seal and spin down briefly. With the SPT Labtech Mosquito or similar liquid handler, use the Tn5 reaction mix plate as the source plate and the L-00X-POOL-1+2-1:100 plate from step 5 as the destination plate, transfer   of Tn5 reaction mix to each well of the destination plate.Immediately seal the destination plate, spin down and place it on the thermocycler. Save the source plate to re-use in step 7.  ProgramTagmentationLid105°CVolume2μL     TempTimeStep 155°C 5minStep 24°Chold While the Tagmentation program is running, start making the source plate for the tagmentation neutralization mixture (you can use the same plate as the Tn5 source plate with different columns).1x Reaction Volume (uL)400X Reaction Volume (uL)Notes Amplicon DNA (1:100 dilution)0.4TAG Buffer (From 2.18X stock)*0.91428512.00Master mix is made for 560 reactions to account for dead volumeN,N-dimethylformamide (DMF) (Sigma-Aldrich, 227056-1L)0.0844.80Homebrew Tn5 † (1:128)0.20571115.20Total1.6672.00More information to test homebrew Tn5 batch: # source wells:16uL/well:38.4 uLPipette into wells A1-P1 of a new 384-well plateBefore making the master mix, make sure the thermocycler is running the Tagmentation program.    In a chemical fume hood, mix DMF and TAG Buffer. Add homebrew recombinant Tn5 to the buffer mix. Move quickly to make the master mix.NOTES:  Tn5 is temperature-sensitive and can precipitate out of solution. Handle with care and avoid multiple freeze-thaw cycles. Stocks of homebrew recombinant Tn5 are diluted in a 50% glycerol mix. It is critical to slowly pipette up and down with a wide-bore or P1000 pipette to thoroughly mix the solution. DO NOT VORTEX. On ice, manually pipette    of the Tn5 master Mix into wells A1-P1 of a Biorad HS 384-well PCR plate. Seal and spin down briefly. With the SPT Labtech Mosquito or similar liquid handler, use the Tn5 reaction mix plate as the source plate and the L-00X-POOL-1+2-1:100 plate from step 5 as the destination plate, transfer   of Tn5 reaction mix to each well of the destination plate.Immediately seal the destination plate, spin down and place it on the thermocycler. Save the source plate to re-use in step 7.  ProgramTagmentationLid105°CVolume2μL     TempTimeStep 155°C 5minStep 24°Chold While the Tagmentation program is running, start making the source plate for the tagmentation neutralization mixture (you can use the same plate as the Tn5 source plate with different columns).In a chemical fume hood, mix DMF and TAG Buffer. Add homebrew recombinant Tn5 to the buffer mix. Move quickly to make the master mix.NOTES:  Tn5 is temperature-sensitive and can precipitate out of solution. Handle with care and avoid multiple freeze-thaw cycles. Stocks of homebrew recombinant Tn5 are diluted in a 50% glycerol mix. It is critical to slowly pipette up and down with a wide-bore or P1000 pipette to thoroughly mix the solution. DO NOT VORTEX. On ice, manually pipette    of the Tn5 master Mix into wells A1-P1 of a Biorad HS 384-well PCR plate. Seal and spin down briefly. With the SPT Labtech Mosquito or similar liquid handler, use the Tn5 reaction mix plate as the source plate and the L-00X-POOL-1+2-1:100 plate from step 5 as the destination plate, transfer   of Tn5 reaction mix to each well of the destination plate.Immediately seal the destination plate, spin down and place it on the thermocycler. Save the source plate to re-use in step 7.  ProgramTagmentationLid105°CVolume2μL     TempTimeStep 155°C 5minStep 24°Chold While the Tagmentation program is running, start making the source plate for the tagmentation neutralization mixture (you can use the same plate as the Tn5 source plate with different columns).NOTES:  Tn5 is temperature-sensitive and can precipitate out of solution. Handle with care and avoid multiple freeze-thaw cycles. Stocks of homebrew recombinant Tn5 are diluted in a 50% glycerol mix. It is critical to slowly pipette up and down with a wide-bore or P1000 pipette to thoroughly mix the solution. DO NOT VORTEX. On ice, manually pipette   of the Tn5 master Mix into wells A1-P1 of a Biorad HS 384-well PCR plate. Seal and spin down briefly.With the SPT Labtech Mosquito or similar liquid handler, use the Tn5 reaction mix plate as the source plate and the L-00X-POOL-1+2-1:100 plate from step 5 as the destination plate, transfer  of Tn5 reaction mix to each well of the destination plate.Immediately seal the destination plate, spin down and place it on the thermocycler. Save the source plate to re-use in step 7.  ProgramTagmentationLid105°CVolume2μL     TempTimeStep 155°C 5minStep 24°Chold While the Tagmentation program is running, start making the source plate for the tagmentation neutralization mixture (you can use the same plate as the Tn5 source plate with different columns).ProgramTagmentationLid105°CVolume2 μLStepsTempTimeStep 155°C 5 minStep 24°CholdTabula Muris Consortium, Shayan Hosseinzadeh(2019).SmartSeq2 for HTP Generation of FACS Sorted Single Cell Libraries.protocols.io https://dx.doi.org/10.17504/protocols.io.2uwgexeHennig, B. P. et al. Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3 Genes Genomes Genetics 8, 79–89 (2018). Picelli, S. et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res 24, 2033–2040 (2014).
Tris-HCl, pH 7.4 (Lonza, 51247)50 mMMgCl2 (Invitrogen, AM9530G)50 mM
1x Reaction Volume (uL)400X Reaction Volume (uL)Notes Amplicon DNA (1:100 dilution)0.4TAG Buffer (From 2.18X stock)*0.91428512.00Master mix is made for 560 reactions to account for dead volumeN,N-dimethylformamide (DMF) (Sigma-Aldrich, 227056-1L)0.0844.80Homebrew Tn5 † (1:128)0.20571115.20Total1.6672.00
# source wells:16uL/well:38.4 uLPipette into wells A1-P1 of a new 384-well plate
38.4 µl
1.2 µl
ProgramTagmentationLid105°CVolume2 μL
StepsTempTimeStep 155°C 5 minStep 24°Chold
?. [Neutralization]
This step is time-sensitive! Tn5 has some activity at room temperature, so move quickly to neutralize the Tn5 reactions. The Tn5 neutralization buffer corresponds to a 0.1% Sodium Dodecyl Sulfate (SDS, Fisher Scientific, BP166-500) solution. Prepare 1% SDS as a stock solution in advance and store at room temperature. Prepare single-use aliquots of the final 0.1% working solution in advance to streamline this step.Pipette the neutralization buffer into wells A3-P3 of the same Biorad HS 384-well PCR plate source plate used in step 6.   Seal the plate, spin down briefly.With the mosquito use this plate as the source plate and the Tn5 reaction plate as the destination plate. Transfer   of the neutralization solution to each well of the Tn5 reaction plate to start inactivation of the tagmentation reaction.Immediately seal the Tn5 reaction plate and centrifuge at full speed for 5 minutes.The Tn5 neutralization buffer corresponds to a 0.1% Sodium Dodecyl Sulfate (SDS, Fisher Scientific, BP166-500) solution. Prepare 1% SDS as a stock solution in advance and store at room temperature. Prepare single-use aliquots of the final 0.1% working solution in advance to streamline this step.Pipette the neutralization buffer into wells A3-P3 of the same Biorad HS 384-well PCR plate source plate used in step 6.   Seal the plate, spin down briefly.With the mosquito use this plate as the source plate and the Tn5 reaction plate as the destination plate. Transfer   of the neutralization solution to each well of the Tn5 reaction plate to start inactivation of the tagmentation reaction.Immediately seal the Tn5 reaction plate and centrifuge at full speed for 5 minutes.Pipette the neutralization buffer into wells A3-P3 of the same Biorad HS 384-well PCR plate source plate used in step 6.   Seal the plate, spin down briefly.With the mosquito use this plate as the source plate and the Tn5 reaction plate as the destination plate. Transfer   of the neutralization solution to each well of the Tn5 reaction plate to start inactivation of the tagmentation reaction.Immediately seal the Tn5 reaction plate and centrifuge at full speed for 5 minutes.Seal the plate, spin down briefly.With the mosquito use this plate as the source plate and the Tn5 reaction plate as the destination plate. Transfer   of the neutralization solution to each well of the Tn5 reaction plate to start inactivation of the tagmentation reaction.Immediately seal the Tn5 reaction plate and centrifuge at full speed for 5 minutes.With the mosquito use this plate as the source plate and the Tn5 reaction plate as the destination plate. Transfer  of the neutralization solution to each well of the Tn5 reaction plate to start inactivation of the tagmentation reaction.
0.4 µl
?. [Index PCR ]
Using the Dragonfly or similar liquid handlers dispense  of NEBNext Ultra II Q5 Master Mix-2X per well of a 384 well Biorad HS PCR plate. You can store this pre-aliquoted single-used plate at -20°C.Start Index PCR program on the thermocycler.Thaw a Nextera dual index barcode plate containing 5 μM stocks of unique i5 and i7 index primers in each well. Briefly spin down at maximum speed. Note: the Q5 plate should thaw quickly. Briefly spin down at maximum speed.Using an Integra or similar liquid handler, stamp 0.8µL from the dual barcode plate to your neutralized amplicon plate.Using the same tips, stamp 2.8 uL from the Q5 plate to the plate with neutralized and cleaned amplicons.  1x Reaction Volume (uL)Tagmented product2Q5 Plate2.8Nextera Dual Index Barcodes0.8Total per well5.6 Seal and spin all plates, making sure that there are no bubbles in the wells. Place the plate on the thermocycler.Thaw a Nextera dual index barcode plate containing 5 μM stocks of unique i5 and i7 index primers in each well. Briefly spin down at maximum speed. Note: the Q5 plate should thaw quickly. Briefly spin down at maximum speed.Using an Integra or similar liquid handler, stamp 0.8µL from the dual barcode plate to your neutralized amplicon plate.Using the same tips, stamp 2.8 uL from the Q5 plate to the plate with neutralized and cleaned amplicons.  1x Reaction Volume (uL)Tagmented product2Q5 Plate2.8Nextera Dual Index Barcodes0.8Total per well5.6 Seal and spin all plates, making sure that there are no bubbles in the wells. Place the plate on the thermocycler. Note: the Q5 plate should thaw quickly. Briefly spin down at maximum speed.Using an Integra or similar liquid handler, stamp 0.8µL from the dual barcode plate to your neutralized amplicon plate.Using the same tips, stamp 2.8 uL from the Q5 plate to the plate with neutralized and cleaned amplicons.  1x Reaction Volume (uL)Tagmented product2Q5 Plate2.8Nextera Dual Index Barcodes0.8Total per well5.6 Seal and spin all plates, making sure that there are no bubbles in the wells. Place the plate on the thermocycler.Using an Integra or similar liquid handler, stamp 0.8µL from the dual barcode plate to your neutralized amplicon plate.Using the same tips, stamp 2.8 uL from the Q5 plate to the plate with neutralized and cleaned amplicons.  1x Reaction Volume (uL)Tagmented product2Q5 Plate2.8Nextera Dual Index Barcodes0.8Total per well5.6 Seal and spin all plates, making sure that there are no bubbles in the wells. Place the plate on the thermocycler.Using the same tips, stamp 2.8 uL from the Q5 plate to the plate with neutralized and cleaned amplicons.  1x Reaction Volume (uL)Tagmented product2Q5 Plate2.8Nextera Dual Index Barcodes0.8Total per well5.6 Seal and spin all plates, making sure that there are no bubbles in the wells. Place the plate on the thermocycler.Reagents1x Reaction Volume (uL)Tagmented product from step 72NEBNext Ultra II Q5 Master Mix (M0544L) (2X)2.8Nextera Dual Index Barcodes (i7 / i5) (5 uM)0.8Total per well5.6ProgramIndex PCRLid105°CVolume4 μLTempTimeCycleStep 172°C 3 min1Step 295°C30 sec1Step 395°C10 sec8Step 455°C30 secStep 572°C1 minStep 672°C5 min1Step 74°Chold1
3.5 µl
Reagents1x Reaction Volume (uL)Tagmented product from step 72NEBNext Ultra II Q5 Master Mix (M0544L) (2X)2.8Nextera Dual Index Barcodes (i7 / i5) (5 uM)0.8Total per well5.6
ProgramIndex PCRLid105°CVolume4 μL
TempTimeCycleStep 172°C 3 min1Step 295°C30 sec1Step 395°C10 sec8Step 455°C30 secStep 572°C1 minStep 672°C5 min1Step 74°Chold1
?. [Library pooling and SPRI cleanup]
Use the mosquito or similar liquid handlers to pool  of the library from each well of the library plate to A1 and A3 columns in a 384 well HS PCR plate.Use a multichannel pipette to pool samples in the A1 and A3 columns to a clean sterile reservoir. Using a P1000 pipette aspirate the whole pooled volume and transfer to a 1.5 ml Eppendorf tube.Use SPRIselect or homebrew SPRI beads to clean the pooled library.Verify the total volume pooled and calculate the required volume of SPRI beads. Add room temperature SPRI beads at a ratio of 0.7X pooled library volume (SPRI beads volume: total pooled library volume) to the PCR enriched/barcoded library reaction. Mix well.Incubate for 5 minutes at room temperature.Place the Eppendorf tube on a magnetic rack and incubate for 5 mins.Remove supernatant. Add   of 80% ethanol to the Eppendorf tube while on the magnetic rack. Incubate at room temperature for 1 min then discard the supernatant.Repeat the ethanol wash step a total of 2 times.Dry the beads for 5 mins while on the magnetic rack.Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding   of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Use SPRIselect or homebrew SPRI beads to clean the pooled library.Verify the total volume pooled and calculate the required volume of SPRI beads. Add room temperature SPRI beads at a ratio of 0.7X pooled library volume (SPRI beads volume: total pooled library volume) to the PCR enriched/barcoded library reaction. Mix well.Incubate for 5 minutes at room temperature.Place the Eppendorf tube on a magnetic rack and incubate for 5 mins.Remove supernatant. Add   of 80% ethanol to the Eppendorf tube while on the magnetic rack. Incubate at room temperature for 1 min then discard the supernatant.Repeat the ethanol wash step a total of 2 times.Dry the beads for 5 mins while on the magnetic rack.Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding   of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Verify the total volume pooled and calculate the required volume of SPRI beads. Add room temperature SPRI beads at a ratio of 0.7X pooled library volume (SPRI beads volume: total pooled library volume) to the PCR enriched/barcoded library reaction. Mix well.Incubate for 5 minutes at room temperature.Place the Eppendorf tube on a magnetic rack and incubate for 5 mins.Remove supernatant. Add   of 80% ethanol to the Eppendorf tube while on the magnetic rack. Incubate at room temperature for 1 min then discard the supernatant.Repeat the ethanol wash step a total of 2 times.Dry the beads for 5 mins while on the magnetic rack.Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding   of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Incubate for 5 minutes at room temperature.Place the Eppendorf tube on a magnetic rack and incubate for 5 mins.Remove supernatant. Add   of 80% ethanol to the Eppendorf tube while on the magnetic rack. Incubate at room temperature for 1 min then discard the supernatant.Repeat the ethanol wash step a total of 2 times.Dry the beads for 5 mins while on the magnetic rack.Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding   of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Place the Eppendorf tube on a magnetic rack and incubate for 5 mins.Remove supernatant. Add   of 80% ethanol to the Eppendorf tube while on the magnetic rack. Incubate at room temperature for 1 min then discard the supernatant.Repeat the ethanol wash step a total of 2 times.Dry the beads for 5 mins while on the magnetic rack.Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding   of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Remove supernatant. Add  of 80% ethanol to the Eppendorf tube while on the magnetic rack. Incubate at room temperature for 1 min then discard the supernatant.Repeat the ethanol wash step a total of 2 times.Dry the beads for 5 mins while on the magnetic rack.Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding   of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Dry the beads for 5 mins while on the magnetic rack.Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding   of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Remove the Eppendorf tube from the magnetic rack. Elute DNA from beads by adding  of nuclease-free water.Vortex to mix. Spin tubes and incubate for 5 mins at room temperature off the magnetic rack.Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Place the Eppendorf tube back on the magnetic rack until the solution is clear ~ 5 min.Remove   of the supernatant and transfer to a clean Eppendorf tube.Check the quality of your clean library on a TapeStation or Bioanalyzer. Remove  of the supernatant and transfer to a clean Eppendorf tube.
2 µl
700 µl
53 µl
50 µl
?. [Quality control]
Quantify library using a fragment analyzer such as TapeStation or BioAnalyzer. Dilute the final library appropriately to fall in the detection range of the assay (HS D5000, D5000, Bioanalyzer). To do this perform an upfront quantification of the cleaned library using the Qubit dsDNA assay or similar.For Agilent D5000 Tapestation assays dilute the final library 1:10 and 1:100.Run undiluted, 1:10, and 1:100 as per assay guidelines on the Agilent Tapestation. Tapestation/Bioanalyzer gives important information about adapter dimers which are seen around the 120-170 bp range. Library peak size should be around 400 bp. additional SPRI cleanup might be necessary to get rid of adapter dimers. The clean amplicon library should be represented by a single peak as shown below. For in-house applications, we measure the region concentration using the Agilent Tapestation. Region concentration is calculated using the area from 200 bp to 1000 bp. From the past 5 library preps, the average region concentration or yield of the library was 38 nmol/L (range: 20.8 - 52.8 nmol/L), and the average library size was 452 bps (range: 425-473 bps).Given samples with a broad range of Ct values (12-30) in a typical library prep, we empirically determined that sequencing 1 million total reads per sample maximized genome recovery yield from 2 x 150 bp paired-end sequencing runs performed on Illumina's NovaSeq 6000, NextSeq 500, or NextSeq 2000.Dilute the final library appropriately to fall in the detection range of the assay (HS D5000, D5000, Bioanalyzer). To do this perform an upfront quantification of the cleaned library using the Qubit dsDNA assay or similar.For Agilent D5000 Tapestation assays dilute the final library 1:10 and 1:100.Run undiluted, 1:10, and 1:100 as per assay guidelines on the Agilent Tapestation. Tapestation/Bioanalyzer gives important information about adapter dimers which are seen around the 120-170 bp range. Library peak size should be around 400 bp. additional SPRI cleanup might be necessary to get rid of adapter dimers. The clean amplicon library should be represented by a single peak as shown below. For in-house applications, we measure the region concentration using the Agilent Tapestation. Region concentration is calculated using the area from 200 bp to 1000 bp. From the past 5 library preps, the average region concentration or yield of the library was 38 nmol/L (range: 20.8 - 52.8 nmol/L), and the average library size was 452 bps (range: 425-473 bps).Given samples with a broad range of Ct values (12-30) in a typical library prep, we empirically determined that sequencing 1 million total reads per sample maximized genome recovery yield from 2 x 150 bp paired-end sequencing runs performed on Illumina's NovaSeq 6000, NextSeq 500, or NextSeq 2000.For Agilent D5000 Tapestation assays dilute the final library 1:10 and 1:100.Run undiluted, 1:10, and 1:100 as per assay guidelines on the Agilent Tapestation. Tapestation/Bioanalyzer gives important information about adapter dimers which are seen around the 120-170 bp range. Library peak size should be around 400 bp. additional SPRI cleanup might be necessary to get rid of adapter dimers. The clean amplicon library should be represented by a single peak as shown below. For in-house applications, we measure the region concentration using the Agilent Tapestation. Region concentration is calculated using the area from 200 bp to 1000 bp. From the past 5 library preps, the average region concentration or yield of the library was 38 nmol/L (range: 20.8 - 52.8 nmol/L), and the average library size was 452 bps (range: 425-473 bps).Given samples with a broad range of Ct values (12-30) in a typical library prep, we empirically determined that sequencing 1 million total reads per sample maximized genome recovery yield from 2 x 150 bp paired-end sequencing runs performed on Illumina's NovaSeq 6000, NextSeq 500, or NextSeq 2000.Run undiluted, 1:10, and 1:100 as per assay guidelines on the Agilent Tapestation. Tapestation/Bioanalyzer gives important information about adapter dimers which are seen around the 120-170 bp range. Library peak size should be around 400 bp. additional SPRI cleanup might be necessary to get rid of adapter dimers. The clean amplicon library should be represented by a single peak as shown below. For in-house applications, we measure the region concentration using the Agilent Tapestation. Region concentration is calculated using the area from 200 bp to 1000 bp. From the past 5 library preps, the average region concentration or yield of the library was 38 nmol/L (range: 20.8 - 52.8 nmol/L), and the average library size was 452 bps (range: 425-473 bps).Given samples with a broad range of Ct values (12-30) in a typical library prep, we empirically determined that sequencing 1 million total reads per sample maximized genome recovery yield from 2 x 150 bp paired-end sequencing runs performed on Illumina's NovaSeq 6000, NextSeq 500, or NextSeq 2000.Tapestation/Bioanalyzer gives important information about adapter dimers which are seen around the 120-170 bp range. Library peak size should be around 400 bp. additional SPRI cleanup might be necessary to get rid of adapter dimers. The clean amplicon library should be represented by a single peak as shown below. For in-house applications, we measure the region concentration using the Agilent Tapestation. Region concentration is calculated using the area from 200 bp to 1000 bp. From the past 5 library preps, the average region concentration or yield of the library was 38 nmol/L (range: 20.8 - 52.8 nmol/L), and the average library size was 452 bps (range: 425-473 bps).Given samples with a broad range of Ct values (12-30) in a typical library prep, we empirically determined that sequencing 1 million total reads per sample maximized genome recovery yield from 2 x 150 bp paired-end sequencing runs performed on Illumina's NovaSeq 6000, NextSeq 500, or NextSeq 2000.For in-house applications, we measure the region concentration using the Agilent Tapestation. Region concentration is calculated using the area from 200 bp to 1000 bp. From the past 5 library preps, the average region concentration or yield of the library was 38 nmol/L (range: 20.8 - 52.8 nmol/L), and the average library size was 452 bps (range: 425-473 bps).Given samples with a broad range of Ct values (12-30) in a typical library prep, we empirically determined that sequencing 1 million total reads per sample maximized genome recovery yield from 2 x 150 bp paired-end sequencing runs performed on Illumina's NovaSeq 6000, NextSeq 500, or NextSeq 2000.From the past 5 library preps, the average region concentration or yield of the library was 38 nmol/L (range: 20.8 - 52.8 nmol/L), and the average library size was 452 bps (range: 425-473 bps).Given samples with a broad range of Ct values (12-30) in a typical library prep, we empirically determined that sequencing 1 million total reads per sample maximized genome recovery yield from 2 x 150 bp paired-end sequencing runs performed on Illumina's NovaSeq 6000, NextSeq 500, or NextSeq 2000.