Aug 14, 2020

Public workspaceIllumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer V.1

DOI
dx.doi.org/10.17504/protocols.io.bcbnisme
  • 1US Food and Drug Administration;
  • 2Food and Drug Administration
  • GenomeTrakr
    Tech. support email: genomeTrakr@fda.hhs.gov
Julie Haendiges
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DOI: dx.doi.org/10.17504/protocols.io.bcbnisme
Protocol Citation: Julie Haendiges, Narjol Gonzalez-Escalona, Ruth Timme, Maria Balkey 2020. Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer. protocols.io https://dx.doi.org/10.17504/protocols.io.bcbnisme
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 07, 2020
Last Modified: November 10, 2021
Protocol Integer ID: 32846
Keywords: Illumina DNA Prep, WGS Library Preparation, GenomeTrakr, Whole Genome Sequencing,
Disclaimer
Please note that this protocol is public domain, which supersedes the CC-BY license default used by protocols.io.
Abstract
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.

This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network.


Complete in order:
1. DNA Extraction (Manual DNA Extraction or Automated DNA Extraction using the Qiacube)
  • Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing

2. DNA Quantitation
  • Quantitation of extracted DNA using the Qubit Flourometer

3. Library Preparation for WGS (Included SOP or Library Preparation using Illumina Nextera XT )
  • Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex)

4. Sequencing using Illumina MiSeq

5. Data Quality Checks and NCBI Submission
Guidelines
Illumina DNA Prep (M) Tagmentation Kit contain 3 components:
Box 1 of 3:
  • SPB (Store at 2-8oC)
  • TSB (Store at room temperature)
  • TWB (Store at room temperature)

Box 2 of 3: (Store at -25 to -15oC)
  • RSB
  • TB1
  • EPM

Box 3 of 3: (Store at 2-8oC)
  • BLT

Abbreviations:
BLT: Bead-Linked Transposome
dsDNA: Double-Stranded DNA
EPM: Enhanced PCR Mix
HT1: Hybridization Buffer
PCR: Polymerase Chain Reaction
PR2: Incorporation Buffer
RSB: Resuspension Buffer
SPB: Sample Purification Beads
TB1: Tagmentation Buffer 1
TSB: Tagment Stop Buffer
TWB: Tagment Wash Buffer
Materials
MATERIALS
ReagentEthanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
ReagentQubit® dsDNA HS assay kit, 100 reactions Life TechnologiesCatalog #Q32851
ReagentNextera DNA CD Indexes (96 samples)Illumina, Inc.Catalog #20018708
ReagentSodium Hydroxide 1NSigma AldrichCatalog #S2770-100ml
ReagentMolecular grade water nuclease-free
ReagentIllumina DNA Prep (M) Tagmentation (96 Samples)Illumina, Inc.Catalog #20018705
ReagentIllumina DNA Prep (M) Tagmentation (24 Samples)Illumina, Inc.Catalog #20018704
Supplies:
  • Qubit Assay Tubes (Thermofisher cat# Q32856)
  • Pipette Tips, sterile, filtered (assorted volumes)
  • Conical Tubes, 10ml and/or 15ml (FisherSci cat# 14-959-53A or equivalent)
  • Solution basins, sterile (FisherSci cat# 13-681-504 or equivalent)
  • 96-well PCR Plates, semi-skirted, flat deck (FisherSci cat# AB-1400L or equivalent)
  • Microcentrifuge tubes, 1.5 ml, sterile (Thermofisher cat# AM12400 or equivalent)
  • Plate Seals (FisherSci cat# AB-0558 or equivalent )

Equipment:
  • Qubit 2.0 or 3.0 Fluorometer
  • Thermocycler
  • Microplate centrifuge
  • Vortex
  • Magnetic Stand-96 (Thermofisher cat# AM10027) (If possible, have two; one for pre-PCR and one for post-PCR)
  • Micropipettes (Single and Multichannel)
  • Ice bucket
  • Microcentrifuge






Safety warnings

Safety information
Chemical Safety Warning: Take proper precautions, and wear appropriate PPE when handling potentially hazardous chemicals. Ensure that chemicals, spent containers, and unused contents are disposed of in accordance with governmental safety standards.

llumina DNA Flex Library Preparation Kit: See Illumina SDSs for additional information. Take proper precautions and wear appropriate PPE when handling reagents.
TSB: GHS Category 1 for eye damage/irritant and is harmful to aquatic life.
TB1: GHS Category 4 for acute toxicity (dust/mist), Category 2A for eye irritant and Category 1B for reproductive toxicity. Contains N,N=Dimethylformamide.
EPM: GHS Category 4 for acute oral toxicity and Category 1 for specific organ toxicity. Contains tetramethylammonium chloride

Before start
Preparation of the Sequencing Workbook: The worksheet is an excel file that can be found in Appendix 1

Prepare the Initial Dilution tab of the DNA Flex Library Prep Workbook as
described below:
Note: The workbook is designed with the following color scheme:
• White fields should be filled in
• Gray fields are optional
• Blue fields contain formulas, which will auto-populate, and should not be altered
1. Enter sample IDsfor all isolates in column A.
2. Designate indices for each sample. Select the set of indices that will be used from the dropdown menu in E2.
3. Enter the concentration of the gDNA in column H.
4. Enter the volume of extracted DNA to be used for library preparation in column J. GenomeTrakr has standardized the starting volume of DNA to 5 μl (gDNA concentration of 20 - 50 ng/μl), however, the recommended quantity of input DNA is 100-500ng. Our recommendation is to use at least 100ng of input DNA. Individual laboratories may adjust input DNA volumes to ensure quantities fall within this range. The recommended minimum volume of input DNA is 2 μl, if DNA is too concentrated, perform a dilution to bring input DNA volume above 2 μl and proceed.
Dilute and Tagment Input DNA
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 the
buffer
Label a 96-well PCR plate with the Run ID.

Add molecular-grade water to the each sample well (from Column K of the workbook)

Add gDNA to the molecular-grade water (per volume in Column J) and mix well by gently pipetting 5 - 10 times.
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
Vortex the TB1 and spin down the tube.
***Scale up this step according to the number of reactions plus 3-4 for dead space volume/error***

Prepare the Tagmentation Master Mix:

Combine Amount10 µL of TB1 with Amount10 µL of BLT

Reagent volumes per sample for tagmentation master mix
Note: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
Vortex the tagmentation master thoroughly to make sure the BLT beads are evenly resuspended in the buffer.
Using fresh tips, transfer Amount20 µL 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

Pipette up and down 10 times mix the 50 μl reaction to resuspend the beads.
Apply an adhesive PCR plate seal to the plate.
Place the plate into the thermocyler and run the tagmentation program.

Program thermocyler to incubate at Temperature55 °C for Duration00:15:00 followed by a Temperature10 °C hold with the lid heated at Temperature100 °C
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.
Upon completion of the incubation, remove the plate from the thermocycler. Proceed to the Post Tagmentation Cleanup step.
Post Tagmentation Clean Up
Post Tagmentation Clean Up
Remove the plate seal.
Add Amount10 µL 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.

Apply an adhesive PCR plate seal to the plate.
Place the plate into the thermocyler and incubate at Temperature37 °C for Duration00:15:00 followed by a Temperature10 °C hold with the lid heated at Temperature100 °C

While samples are incubating, thaw EPM (stored in freezer) on ice and thaw indices at room temperature.
Remove the plate from the thermocycler, quick spin the plate and remove the seal.
Place the plate on a magnet for Duration00:03:00 or until solution is clear.

Note: The DNA is tagged with adapters and bound to the beads.

Using a multichannel pipette, remove the supernatant and discard.
Remove the plate from the magnet and add Amount100 µL of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended, try to avoid creation of foam from TWB.

Place the plate on the magnet for Duration00:03:00 or until solution is clear.

Remove the supernatant and discard.
Remove the plate from the magnet and add Amount100 µL of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.

Place the plate on the magnet for Duration00:03:00 or until solution is clear.
Remove the supernatant and discard.
Remove the plate from the magnet and add Amount100 µL of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.

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
Amplification and Index Addition of Tagmented DNA
Invert the EPM to mix, then briefly centrifuge.
Briefly centrifuge the Index plate.
***Scale up this step according to the number of reactions plus 3-4 for dead space volume/error***

Prepare the PCR master mix:

Combine Amount20 µL of EPM with Amount20 µL of Molecular grade water

Reagent volumes per sample for PCR master mix
Nte: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
Vortex and spin down the PCR master mix.
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.
Remove the plate from the magnet and immediately proceed to adding the master mix.
Add Amount40 µL 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

Add Amount10 µL 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.

Use a pipette to gently mix a minimum of 10 times to ensure thorough mixing.
Apply an adhesive PCR plate seal to the plate.
Place the plate into the thermocycler and run the following pre-programmed settings with a heated lid at Temperature100 °C

Thermocycler protocol (for use with DNA inputs above 100ng)
Step 1: Temperature68 °C for Duration00:03:00
Step 2: Temperature98 °C for Duration00:03:00
Step 3: 5 cycles of:
Temperature98 °C for Duration00:00:45
Temperature62 °C for Duration00:00:30
Temperature68 °C for Duration00:02:00
Step 4: Temperature68 °C for Duration00:01:00
Step 5: Hold at Temperature10 °C

Centrifuge plate at Centrifigation280 x g for Duration00:01:00

This is a safe stopping point. The plate may be sealed and stored at Temperature2 °C to
Temperature8 °C for up to 3 days.

Pause
Clean up Libraries
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.
Before starting, prepare reagents:
Prepare fresh 80% ethanol sufficient for all samples.
Bring RSB to room temperature (from freezer) and vortex to mix.
Bring SPB to room temperature (at least 30 minutes) from refrigerator. Vortex and invert SPB several times to full resuspend the beads.
Prepare the SPB Master mix:

Note: The ration 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.
If plate was retrieved from 4oC storage, centrifuge plate at 280 x g for 1 minute.

Remove the seal.
Place the sample plate on the magnet for Duration00:05:00

Transfer Amount45 µL of supernatant (now containing the DNA) to a set of new wells on the sample plate.

Remove sample plate from the magnet.
Vortex SPB master mix thoroughly and add Amount85 µL to each PCR product.

Note: The master mix can be added to a reagent basin and distributed using a multichannel pipet
Pipette to mix a minimum of 10 times or until thoroughly mixed.
Incubate at room temperature for Duration00:05:00
Place the 96-well plate on the magnet for Duration00:05:00 or until supernatant is clear.

During incubation, vortex the stock SPB to resuspend the beads.

With the plate still on the magnet, transfer Amount125 µL of supernatant (containing the DNA) to a new set of wells.

Remove the plate from the magnet and add Amount15 µL of stock SPB to the supernatant.

Gently pipet at least 10 times to mix.
Incubate at room temperature for Duration00:05:00

Place on the magnet for Duration00:05:00 or until clear.

Remove and discard the supernatant (DNA is now bound to the beads) without disrupting the beads
Perform the steps below twice (for a total of two washes)
While the plate is on the magnet, add Amount180 µL of the prepared 80% ethanol


Note: Do not add directly to the bead and do not mix.
Incubate for Duration00:00:30

Remove and discard ethanol.
Use a pipette to remove any excess liquid from the plate.

Use a small volume pipette to get out any residual if necessary.
Allow beads to air dry for up to Duration00:05:00 (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.
Remove the plate from the magnet and add Amount32 µL of RSB. Pipet thoroughly to mix

Incubate at room temperature for Duration00:02:00
Place the plate on the magnet for Duration00:02:00
Prepare a new 96-well plate as in below that is labelled with run date and intials. TransferAmount25 µL of the supernatant to a new 96-well plate. This is the final library.


Example 96-well plate set up

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 Temperature-20 °C for up to 30 days.
Pause
Quantification, Normalization and Pooling of Libraries
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.
Enter the Qubit values into the “Normalization and Pooling” tab on the DNA Flexwork sheet. The DNA Flex worksheet can be found in section 8 of this SOP. The dilution values will be calculated automatically.

For v2 sequencing chemistry, it is recommended to dilute to 2nm; and for v3 sequencing chemistry to dilute to 4nm. This value can be adjusted by the dropdown cell H4.
Dilute each sample according to the values on the worksheet in the designated dilution wells on the 96-well plate.
Pool Amount5 µL of each diluted library into the specified well and pipet to mix.

Denaturing Pooled LIbrary
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.


Transfer Amount5 µL of the diluted library to a new Eppendorf LoBind tube.

Add Amount5 µL of 0.2N NaOH and pipette to mix.

Incubate at room temperature for Duration00:05:00 to denature the dsDNA.

Immediately add Amount990 µL of HT1 and pipette to mix. The concentration for a 2 nm start is 10 pM and for 4 nm is 20 pM.

Dilute the denatured library to the final desired loading concentration.
Note: The final loading concentration for optimal Cluster Density may need to be adjusted based on data from previous runs.

Mix by repeated inversion of sample tube.

Optional: Denatured PhiX control can be spiked in at this point.
Heat the denatured DNA library to Temperature96 °C for Duration00:02:00 in a heat block to ensure complete denaturation of all dsDNA in the sample.

Immediately cool in an ice-water bath for at least Duration00:05:00 prior to loading.

The DNA library may sit on ice or at Temperature2 °C - Temperature8 °C until ready for loading (<30 minutes)

Proceed to loading Amount600 µL of the denatured ibrary to the thawed cartridge.

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
Appendix 1

DNA Flex Library Prepartion Workbook

Download GT_LIBRARYPREP_Appendix 1 DNAFlex Worksheet.xlsxGT_LIBRARYPREP_Appendix 1 DNAFlex Worksheet.xlsx

Appendix 2

Nextera DNA CD Indexes (96 indexes, plated)