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]
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

[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
                        • Blue fields contain formulas, which will auto-populate, and should not be altered
1. Enter sample IDs for all isolates in column B.
2. Enter the organism in column C. 
3. Enter the genome size in column D based on the table below: 

 
4. Confirm that the number of isolates on the run is appropriate for the capacity of the reagent kit to be used. The sum of the genome sizes (Mb) for all samples on the run will be displayed in the workbook (Cell D43). This should not exceed the recommended DNA load values for the reagent kit. 

 

5. Enter the Qubit concentration of the gDNA in column E.
6. Determine which index kit will be used and select from the drop-down menu in column I. Select the index well positions in column J. 
7. Enter the volume of extracted DNA to be used for library preparation in column F. 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 100-500 ng. Our recommendation is to use at least 100 ng 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.

[GUIDELINES]
Illumina DNA Prep (M) Tagmentation Kit: The components of the kit have different storage conditions, please follow these recommendations.

Room Temperature Storage:
   IPB (Store upright)
   TSB 
   TWB

 -25 to -15ºC Storage:
   RSB
   TB1
   EPM

 2-8ºC Storage:
   BLT (Store upright)

Abbreviations:
BLT: Bead-Linked Transposome
CD: Combinatorial Dual
dsDNA: Double-Stranded DNA
EPM: Enhanced PCR Mix
HT1: Hybridization Buffer
IPB: Illumina Purification Beads
PCR: Polymerase Chain Reaction
RSB: Resuspension Buffer
TB1: Tagmentation Buffer 1
TSB: Tagment Stop Buffer
TWB: Tagment Wash Buffer
UD: Unique Dual


[STEPS]
SECTION: Dilute and Tagment Input DNA
1. 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
SECTION: Dilute and Tagment Input DNA
2. Label a 96-well PCR plate with the Run ID.

SECTION: Dilute and Tagment Input DNA
3. Add molecular-grade water to the each sample well (from Column G of the workbook)

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

Prepare the Tagmentation Master Mix: 

Combine 10 µL  of TB1 with 10 µL of BLT 

 
Note: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
SECTION: Dilute and Tagment Input DNA
8. Vortex the Tagmentation Master Mix thoroughly to make sure the BLT beads are evenly resuspended in the buffer.
SECTION: Dilute and Tagment Input DNA
9. Using fresh tips, transfer 20 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

SECTION: Dilute and Tagment Input DNA
10. Pipette up and down 10 times mix the 50 μl reaction to resuspend the beads.
SECTION: Dilute and Tagment Input DNA
11. Apply an adhesive PCR plate seal to the plate.
SECTION: Dilute and Tagment Input DNA
12. Place the plate into the thermocyler and run the tagmentation program.

Program thermocyler to incubate  at 55 °C for 15 min  followed by a 10 °C hold with the lid heated at 100 °C
SECTION: Dilute and Tagment Input DNA
13. 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.
SECTION: Dilute and Tagment Input DNA
14. Upon completion of the incubation, remove the plate from the thermocycler. Proceed to the Post Tagmentation Cleanup step.
SECTION: Post Tagmentation Clean Up
15. Remove the plate seal.
SECTION: Post Tagmentation Clean Up
16. Add 10 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.

SECTION: Post Tagmentation Clean Up
17. Apply an adhesive PCR plate seal to the plate.
SECTION: Post Tagmentation Clean Up
18. Place the plate into the thermocyler and incubate at 37 °C for 15 min followed by a 10 °C hold with the lid heated at 100 °C 

SECTION: Post Tagmentation Clean Up
19. While samples are incubating, thaw EPM on ice and thaw indices at room temperature.
SECTION: Post Tagmentation Clean Up
20. Remove the plate from the thermocycler, quick spin the plate and remove the seal.
SECTION: Post Tagmentation Clean Up
21. Place the plate on a magnet for 3 min or until solution is clear.

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

SECTION: Post Tagmentation Clean Up
22. Using a multichannel pipette, remove the supernatant and discard.
SECTION: Post Tagmentation Clean Up
23. Remove the plate from the magnet and add 100 µL of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended, try to avoid creation of foam from TWB.

SECTION: Post Tagmentation Clean Up
24. Place the plate on the magnet for 3 min or until solution is clear.

SECTION: Post Tagmentation Clean Up
25. Remove the supernatant and discard.
SECTION: Post Tagmentation Clean Up
26. Remove the plate from the magnet and add 100 µL of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.

SECTION: Post Tagmentation Clean Up
27. Place the plate on the magnet for 3 min or until solution is clear.
SECTION: Post Tagmentation Clean Up
28. Remove the supernatant and discard.
SECTION: Post Tagmentation Clean Up
29. Remove the plate from the magnet and add 100 µL of TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.

SECTION: Post Tagmentation Clean Up
30. 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.
SECTION: Amplification of Tagmented DNA and Index Addition
31. Invert the EPM to mix, then briefly centrifuge.
SECTION: Amplification of Tagmented DNA and Index Addition
32. Briefly centrifuge the Index plate.
SECTION: Amplification of Tagmented DNA and Index Addition
33. ***Scale up this step according to the number of reactions plus 3-4 for dead space volume/error***

Prepare the PCR master mix:

Combine 20 µL of EPM with 20 µL of Molecular grade water

 
Note: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
SECTION: Amplification of Tagmented DNA and Index Addition
34. Vortex and spin down the PCR master mix.
SECTION: Amplification of Tagmented DNA and Index Addition
35. 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.
SECTION: Amplification of Tagmented DNA and Index Addition
36. Remove the plate from the magnet and immediately proceed to adding the master mix.
SECTION: Amplification of Tagmented DNA and Index Addition
37. Add 40 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

SECTION: Amplification of Tagmented DNA and Index Addition
38. Add 10 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.

SECTION: Amplification of Tagmented DNA and Index Addition
39. Use a pipette to gently mix a minimum of 10 times to ensure thorough mixing.
SECTION: Amplification of Tagmented DNA and Index Addition
40. Apply an adhesive PCR plate seal to the plate.
SECTION: Amplification of Tagmented DNA and Index Addition
41. Place the plate into the thermocycler and run the following pre-programmed settings with a heated lid at 100 °C 

    Thermocycler protocol (for use with DNA inputs above 100ng)
            Step 1: 68 °C  for 3 min 
            Step 2: 98 °C  for 3 min 
            Step 3: 5 cycles of:
                    98 °C  for 45 s 
                    62 °C  for 30 s 
                    68 °C  for 2 min 
            Step 4: 68 °C  for 1 min 
            Step 5: Hold at 10 °C 

SECTION: Amplification of Tagmented DNA and Index Addition
42. Centrifuge plate at 280 x g for 1 min 

SECTION: Amplification of Tagmented DNA and Index Addition
43. This is a safe stopping point. The plate may be sealed and stored at 2 °C to 
8 °C for up to 3 days.

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
44. 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.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
45. Before starting, prepare reagents: 
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
45.1. Prepare fresh 80% ethanol sufficient for all samples. 
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
45.2. Bring RSB to room temperature (from freezer) and vortex to mix.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
45.3. Vortex and invert IPB (stored at room temperature) several times to full resuspend the beads.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
48. Pipette 40 µL of molecular grade water to enough wells on either a new 96-well plate or a set of new wells on the sample plate. 


SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
46. If plate was retrieved from 4ºC storage, centrifuge plate at 280 x g for 1 minute.

Remove the seal.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
47. Place the sample plate on the magnet for 5 min 

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
49. Transfer 45 µL of the supernatant (now containing the DNA) to the corresponding wells containing the 40 μl of water. 

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
50. Remove sample plate from the magnet. 
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
51. Vortex IPB  thoroughly and add 45 µL to each well containing PCR product and water. 

Note: The IPB can be added to a reagent basin and distributed using a multichannel pipet. 

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. 
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
52. Pipette to mix a minimum of 10 times or until thoroughly mixed.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
53. Incubate at room temperature for 5 min
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
54. Place the 96-well plate on the magnet for 5 min or until supernatant is clear.

During incubation, vortex the IPB to resuspend the beads.

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
55. With the plate still on the magnet, transfer 125 µL of supernatant (containing the DNA) to a new set of wells. 

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
56. Remove the plate from the magnet and add 15 µL of IPB to the supernatant. 

Note: The IPB can be added to a reagent basin and distributed using a multichannel pipet. 

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
57. Gently pipet at least 10 times to mix. 
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
58. Incubate at room temperature for 5 min

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
59. Place on the magnet for 5 min or until clear.

Remove and discard the supernatant (DNA is now bound to the beads) without disrupting the beads
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
60. Perform the steps below twice (for a total of two washes)
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
60.1. While the plate is on the magnet, add 180 µL of the prepared 80% ethanol


Note: Do not add directly to the bead and do not mix.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
60.2. Incubate for 30 s

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
60.3. Remove and discard ethanol.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
61. Use a pipette to remove any excess liquid from the plate.

Use a small volume pipette to get out any residual if necessary.
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
62. Allow beads to air dry for up to  5 min (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. 
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
63. Remove the plate from the magnet and add 32 µL of RSB.  Pipet thoroughly to mix

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
64. Incubate at room temperature for 2 min
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
65. Place the plate on the magnet for 2 min
SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
66. Prepare a new 96-well plate as in below that is labelled with run date and intials. Transfer25 µL of the supernatant to a new 96-well plate. This is the final library.


 
 

SECTION: Dual Size Selection and Post-PCR Cleanup of Libraries
67. 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 -20 °C

Note: Setting up your libraries in this fashion will allow for repeat runs if necessary. The libraries are stable for at least 3 months stored at -20 ºC (beyond that time has not been validated by FDA).
SECTION: Quantification, Normalization and Pooling of Libraries
68. 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.
SECTION: Quantification, Normalization and Pooling of Libraries
69. Enter the Qubit values into the "Post-Library" section on the DNA Prep Library worksheet (Column K). The DNA Prep worksheet can be found in section 8 of this SOP. 

SECTION: Quantification, Normalization and Pooling of Libraries
70. For v2 sequencing chemistry, it is recommended to dilute to 2nm; and for v3 sequencing chemistry to dilute to 4nm. Enter the correct value (2 or 4) in Column M. 
SECTION: Quantification, Normalization and Pooling of Libraries
71. The dilution values will auto populate for a 15 μl volume. This value can be changed in Column N if needed by the individual labs. 

Dilute each sample according to the values on the worksheet (Column O and P) in the designated dilution wells on the 96-well plate. 

Note: Resuspension Buffer, Molecular Grade Water, or 10mmTris-HCl with 0.1% Tween20 can be used as diluents.
SECTION: Quantification, Normalization and Pooling of Libraries
72. Pool 5 µL of each diluted library into the specified well and pipet to mix.

 Note: if sequencing Campylobacter, pool 2.5 µL of diluted sample.

SECTION: Denaturing Pooled LIbrary
73. 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. 


SECTION: Denaturing Pooled LIbrary
74. Transfer 5 µL of the diluted library to a new Eppendorf LoBind tube.

SECTION: Denaturing Pooled LIbrary
75. Add 5 µL of 0.2N NaOH and pipette to mix.

SECTION: Denaturing Pooled LIbrary
76. Incubate at room temperature for 5 min to denature the dsDNA. 

SECTION: Denaturing Pooled LIbrary
77. Immediately add 990 µLof HT1 and pipette to mix. The concentration for a 2 nm start is 10 pM and for 4 nm is 20 pM.

SECTION: Denaturing Pooled LIbrary
78. Dilute the denatured library to the final desired loading concentration.  
 

SECTION: Denaturing Pooled LIbrary
79. Mix by repeated inversion of sample tube. 

Optional: Denatured PhiX control can be spiked in at this point. 
SECTION: Denaturing Pooled LIbrary
80. Heat the denatured DNA library to 96 °C  for 2 min in a heat block to ensure complete denaturation of all dsDNA in the sample. 

SECTION: Denaturing Pooled LIbrary
81. Immediately cool in an ice-water bath for at least 5 min prior to loading. 

The DNA library may sit on ice or at 2 °C - 8 °C until ready for loading (<30 minutes)

SECTION: Denaturing Pooled LIbrary
82. Proceed to loading 600 µL of the denatured ibrary to the thawed cartridge.

SECTION: Denaturing Pooled LIbrary
83. Illumina MiSeq Operation and Maintenance: See “Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing” SOP for further instructions.
SECTION: Appendix
84. Appendix 1

DNA Prep Library Workbook
  

SECTION: Appendix
85. Resources:

Illumina DNA Prep Reference Guide