TITLE: Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer
AUTHORS: Julie Haendiges, Narjol Gonzalez-Escalona, Ruth Timme, Maria Balkey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This procedure outlines the protocol for whole genome sequencing of bacterial organisms using the Illumina DNA Prep library preparation kit for sequencing on an Illumina MiSeq sequencer. </div><br/><div class = "text-block">This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network.</div><br/><br/><div class = "text-block"><span style = "font-weight:bold;">Complete in order:</span></div><div class = "text-block"><span style = "font-weight:bold;">1. DNA Extraction (Manual DNA Extraction </span><span style = "font-weight:bold;font-style:italic;">or</span><span style = "font-weight:bold;"> Automated DNA Extraction using the Qiacube)</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">2. DNA Quantitation</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS (Included SOP </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><br/><div class = "text-block"><span style = "font-weight:bold;">2. DNA Quantitation</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS (Included SOP </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><br/><div class = "text-block"><span style = "font-weight:bold;">3. Library Preparation for WGS (Included SOP </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><br/><div class = "text-block"><span style = "font-weight:bold;">4. Sequencing using Illumina MiSeq</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">5. Data Quality Checks and NCBI Submission</span></div></div>

[STEPS]
?. [Dilute and Tagment Input DNA]
Bring BLT (stored in refrigerator)  and TB1 (stored in freezer) to room temperature. Ensure that BLT is stored upright at all times, so that the beads remain submerged in thebuffer
?. [Dilute and Tagment Input DNA]
Label a 96-well PCR plate with the Run ID.
?. [Dilute and Tagment Input DNA]
Add molecular-grade water to the each sample well (from Column K of the workbook)
?. [Dilute and Tagment Input DNA]
Add gDNA to the molecular-grade water (per volume in Column J) and mix well by gently pipetting 5 - 10 times.
?. [Dilute and Tagment Input DNA]
Vortex BLT vigorously for 10 seconds, visually check the beads for complete resuspension and repeat vortexing if necessary. Do not spin down the BLT tube, the beads must be resuspended
?. [Dilute and Tagment Input DNA]
Vortex the TB1 and spin down the tube.
?. [Dilute and Tagment Input DNA]
***Scale up this step according to the number of reactions plus 3-4 for dead space volume/error***Prepare the Tagmentation Master Mix: Combine   of TB1 with  of BLT Note: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
10 µl
10 µl
?. [Dilute and Tagment Input DNA]
Vortex the tagmentation master thoroughly to make sure the BLT beads are evenly resuspended in the buffer.
?. [Dilute and Tagment Input DNA]
Using fresh tips, transfer  of tagmentation master mix to each sample well. Note: The master mix can be added to a reagent basin and distributed using a multichannel pipet
20 µl
?. [Dilute and Tagment Input DNA]
Pipette up and down 10 times mix the 50 μl reaction to resuspend the beads.
?. [Dilute and Tagment Input DNA]
Apply an adhesive PCR plate seal to the plate.
?. [Dilute and Tagment Input DNA]
Place the plate into the thermocyler and run the tagmentation program.Program thermocyler to incubate  at  for   followed by a  hold with the lid heated at
55 °C
10 °C
100 °C
?. [Dilute and Tagment Input DNA]
Check TSB for precipitate (if present, warm at 37 oC for up to 10 minutes and vortex) and ensure it is at room temperature prior to use.
?. [Dilute and Tagment Input DNA]
Upon completion of the incubation, remove the plate from the thermocycler. Proceed to the Post Tagmentation Cleanup step.
?. [Post Tagmentation Clean Up ]
Remove the plate seal.
?. [Post Tagmentation Clean Up ]
Add  of TSB to each sample ( A multi-channel pipette can be used) Gently pipette up and down 10 times to mix and fully resuspend the beads in the 50 μl reaction.
10 µl
?. [Post Tagmentation Clean Up ]
Apply an adhesive PCR plate seal to the plate.
?. [Post Tagmentation Clean Up ]
Place the plate into the thermocyler and incubate at  for  followed by a  hold with the lid heated at
37 °C
10 °C
100 °C
?. [Post Tagmentation Clean Up ]
While samples are incubating, thaw EPM (stored in freezer) on ice and thaw indices at room temperature.
?. [Post Tagmentation Clean Up ]
Remove the plate from the thermocycler, quick spin the plate and remove the seal.
?. [Post Tagmentation Clean Up ]
Place the plate on a magnet for  or until solution is clear.Note: The DNA is tagged with adapters and bound to the beads.
?. [Post Tagmentation Clean Up ]
Using a multichannel pipette, remove the supernatant and discard.
?. [Post Tagmentation Clean Up ]
Remove the plate from the magnet and add of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended, try to avoid creation of foam from TWB.
100 µl
?. [Post Tagmentation Clean Up ]
Place the plate on the magnet for  or until solution is clear.
?. [Post Tagmentation Clean Up ]
Remove the supernatant and discard.
?. [Post Tagmentation Clean Up ]
Remove the plate from the magnet and add of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.
100 µl
?. [Post Tagmentation Clean Up ]
Place the plate on the magnet for  or until solution is clear.
?. [Post Tagmentation Clean Up ]
Remove the supernatant and discard.
?. [Post Tagmentation Clean Up ]
Remove the plate from the magnet and add of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.
100 µl
?. [Post Tagmentation Clean Up ]
Place the plate with TWB on the magnet and allow to incubate until ready to proceed with adding the PCR master mix in the Amplify Tagmented DNA step. The plate should incubate for at least 3 minutes. It is important to keep the pellet in TWB to prevent overdrying of the beads.
?. [Amplification and Index Addition of Tagmented DNA]
Invert the EPM to mix, then briefly centrifuge.
?. [Amplification and Index Addition of Tagmented DNA]
Briefly centrifuge the Index plate.
?. [Amplification and Index Addition of Tagmented DNA]
***Scale up this step according to the number of reactions plus 3-4 for dead space volume/error***Prepare the PCR master mix:Combine  of EPM with  of Molecular grade waterNote: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
20 µl
20 µl
?. [Amplification and Index Addition of Tagmented DNA]
Vortex and spin down the PCR master mix.
?. [Amplification and Index Addition of Tagmented DNA]
Remove the third TWB wash from the samples while on the magnet. Remove any excess liquid from the plate using a small volume pipette.Note: Removal of TWB is crucial, as it can impede PCR. Any foam remaining on the wells will not negatively impact the library.
?. [Amplification and Index Addition of Tagmented DNA]
Remove the plate from the magnet and immediately proceed to adding the master mix.
?. [Amplification and Index Addition of Tagmented DNA]
Add  of PCR master mix to each sample well. Gently pipette to mix to ensure beads are resuspended.Note: The master mix can be added to a reagent basin and distributed using a multichannel pipet
40 µl
?. [Amplification and Index Addition of Tagmented DNA]
Add  of the index primer pair from the appropriate index wells in accordance with the sample sheet. The plate has a foil seal on it, P20 tips are sufficient to pierce the seal to pipette. The indexes are single-use only.
10 µl
?. [Amplification and Index Addition of Tagmented DNA]
Use a pipette to gently mix a minimum of 10 times to ensure thorough mixing.
?. [Amplification and Index Addition of Tagmented DNA]
Apply an adhesive PCR plate seal to the plate.
?. [Amplification and Index Addition of Tagmented DNA]
Place the plate into the thermocycler and run the following pre-programmed settings with a heated lid at     Thermocycler protocol (for use with DNA inputs above 100ng)            Step 1:   for             Step 2:   for             Step 3: 5 cycles of:  for   for   for             Step 4:   for             Step 5: Hold at
100 °C
68 °C
98 °C
98 °C
62 °C
68 °C
68 °C
10 °C
?. [Amplification and Index Addition of Tagmented DNA]
Centrifuge plate at  for
Centrifuge: 280 34
?. [Amplification and Index Addition of Tagmented DNA]
This is a safe stopping point. The plate may be sealed and stored at  to  for up to 3 days.
2 °C
8 °C
?. [Clean up Libraries]
NOTE: The steps listed below are critical for efficient size selection, product recovery and thus cluster generation and sequencing. Always check pipette tips for correct volumes and ensure that no beads have accidentally been aspirated. If beads have been aspirated or the bead pellet is disturbed, allow the pellet to reform (3-5 minutes on the magnet) and repeat the step.
?. [Clean up Libraries]
Before starting, prepare reagents:
?. [Clean up Libraries]
Prepare fresh 80% ethanol sufficient for all samples.
?. [Clean up Libraries]
Bring RSB to room temperature (from freezer) and vortex to mix.
?. [Clean up Libraries]
Bring SPB to room temperature (at least 30 minutes) from refrigerator. Vortex and invert SPB several times to full resuspend the beads.
?. [Clean up Libraries]
Prepare the SPB Master mix:Note: The ratio of SPB:water has been validated by FDA-CFSAN for size-selection.  CDC PulseNet uses a different ratio of SPB:water to select for large insert sizes.
?. [Clean up Libraries]
If plate was retrieved from 4oC storage, centrifuge plate at 280 x g for 1 minute.Remove the seal.
?. [Clean up Libraries]
Place the sample plate on the magnet for
?. [Clean up Libraries]
Transfer of supernatant (now containing the DNA) to a set of new wells on the sample plate.
45 µl
?. [Clean up Libraries]
Remove sample plate from the magnet.
?. [Clean up Libraries]
Vortex SPB master mix thoroughly and add  to each PCR product. Note: The master mix can be added to a reagent basin and distributed using a multichannel pipet
85 µl
?. [Clean up Libraries]
Pipette to mix a minimum of 10 times or until thoroughly mixed.
?. [Clean up Libraries]
Incubate at room temperature for
?. [Clean up Libraries]
Place the 96-well plate on the magnet for  or until supernatant is clear.During incubation, vortex the stock SPB to resuspend the beads.
?. [Clean up Libraries]
With the plate still on the magnet, transfer  of supernatant (containing the DNA) to a new set of wells.
125 µl
?. [Clean up Libraries]
Remove the plate from the magnet and add  of stock SPB to the supernatant.
15 µl
?. [Clean up Libraries]
Gently pipet at least 10 times to mix.
?. [Clean up Libraries]
Incubate at room temperature for
?. [Clean up Libraries]
Place on the magnet for  or until clear.Remove and discard the supernatant (DNA is now bound to the beads) without disrupting the beads
?. [Clean up Libraries]
Perform the steps below twice (for a total of two washes)
?. [Clean up Libraries]
While the plate is on the magnet, add  of the prepared 80% ethanolNote: Do not add directly to the bead and do not mix.
180 µl
?. [Clean up Libraries]
Incubate for
?. [Clean up Libraries]
Remove and discard ethanol.
?. [Clean up Libraries]
Use a pipette to remove any excess liquid from the plate.Use a small volume pipette to get out any residual if necessary.
?. [Clean up Libraries]
Allow beads to air dry for up to   (minimum of 3 minutes)Note: Do not allow beads to over-dry. If bead pellet appears to be cracking, immediately resuspend beads regardless of drying time.
?. [Clean up Libraries]
Remove the plate from the magnet and add  of RSB.  Pipet thoroughly to mix
32 µl
?. [Clean up Libraries]
Incubate at room temperature for
?. [Clean up Libraries]
Place the plate on the magnet for
?. [Clean up Libraries]
Prepare a new 96-well plate as in below that is labelled with run date and intials. Transfer of the supernatant to a new 96-well plate. This is the final library.
25 µl
?. [Clean up Libraries]
If ready to proceed go to the Pooling Libraries step. Otherwise, this is a safe stopping point. The plate may be sealed and stored at  for up to 30 days.
-20 °C
?. [Quantification, Normalization and Pooling of Libraries]
Quantify each sample using the Qubit dsDNA High Sensitivity kit. See SOP titled“DNA Quantification using the Qubit Fluorometer”  for more detailed information on performing DNA quantification.
?. [Quantification, Normalization and Pooling of Libraries]
Enter the Qubit values into the "Prep Workbook" tab on the DNA Prep Library worksheet. The DNA Prep worksheet can be found in section 8 of this SOP. The dilution values will be calculated automatically.
?. [Quantification, Normalization and Pooling of Libraries]
For v2 sequencing chemistry, it is recommended to dilute to 2nm; and for v3 sequencing chemistry to dilute to 4nm.
?. [Quantification, Normalization and Pooling of Libraries]
Dilute each sample according to the values on the worksheet in the designated dilution wells on the 96-well plate.
?. [Quantification, Normalization and Pooling of Libraries]
Pool  of each diluted library into the specified well and pipet to mix. Note: if sequencing Campylobacter, pool  of diluted sample.
5 µl
2.5 µl
?. [Denaturing Pooled LIbrary]
Prepare a fresh aliquot of 0.2N NaOH. (This should be made fresh for each run)Note: It is recommended to make aliquots of 1N NaOH and store in the freezer.
?. [Denaturing Pooled LIbrary]
Transfer  of the diluted library to a new Eppendorf LoBind tube.
5 µl
?. [Denaturing Pooled LIbrary]
Add  of 0.2N NaOH and pipette to mix.
5 µl
?. [Denaturing Pooled LIbrary]
Incubate at room temperature for  to denature the dsDNA.
?. [Denaturing Pooled LIbrary]
Immediately add of HT1 and pipette to mix. The concentration for a 2 nm start is 10 pM and for 4 nm is 20 pM.
990 µl
?. [Denaturing Pooled LIbrary]
Dilute the denatured library to the final desired loading concentration.
?. [Denaturing Pooled LIbrary]
Mix by repeated inversion of sample tube. Optional: Denatured PhiX control can be spiked in at this point.
?. [Denaturing Pooled LIbrary]
Heat the denatured DNA library to   for  in a heat block to ensure complete denaturation of all dsDNA in the sample.
96 °C
?. [Denaturing Pooled LIbrary]
Immediately cool in an ice-water bath for at least  prior to loading. The DNA library may sit on ice or at  -  until ready for loading (
2 °C
8 °C
?. [Denaturing Pooled LIbrary]
Proceed to loading  of the denatured ibrary to the thawed cartridge.
600 µl
?. [Denaturing Pooled LIbrary]
Illumina MiSeq Operation and Maintenance: See “Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing” SOP for further instructions.
?. [Appendix]
Appendix 1DNA Prep Library Workbook
?. [Appendix]
Appendix 2Nextera DNA CD Indexes (96 indexes, plated)