Chapter 7 Library indexing

The second step of the adaptor-based library preparation is to amplify the library using primers containing unique identifiers. This step serves the double function of increasing the molarity of the library to high-enough levels for sequencing, and to assign a sample-specific molecular tag to each library. As library preparation efficiency can be very variable when working with a diverge range of samples derived from different taxa, in the EHI pipeline we implement a qPCR screening that inform us about the optimal number of PCR cycles the libraries should be subject to for optimal library preparation.

It is crucial to carefully adjust the number of indexing PCR cycles to prevent the over-amplification of libraries, which can lead to the generation of highly clonal libraries. With each PCR cycle, an identical copy of an existing DNA sequence is produced. When libraries are excessively amplified, the resultant library could be predominantly composed of technical PCR duplicates rather than the original DNA sequence templates. Failure to appropriately calibrate this step might lead to sequencing only a fraction of the original sample’s complexity. This outcome could artificially distort the diversity of DNA molecules, potentially resulting in erroneous data interpretations.

7.1 Instruments, plasticware and reagents

Instruments

  • Real Time PCR (qPCR) Instrument such as Mx3000 qPCR System (Agilent)
  • Agarose Gel Electrophoresis System
  • Thermocycler for amplification
  • Magnet compatible with 96-well plates such as 96S Super Magnet (Alpaca, SKU: A001322)
  • Fragment analysis system such as 5300 Fragment Analyzer system (Agilent) or 2100 Bioanalyzer (Agilent)
  • SAFE® Screw Cap De-/Capper (LVL technogies)
  • SAFE® 2D/1D Code Reader (LVL technogies)

Plasticware

Item Brand Catalogue number
qPCR Strips EU 8-tube strip, 0.1 mL (for qPCR) BioPlastics B72711
qPCR lids EU Optical Wide area 8-Cap Strip BioPlastics B57801
PCR tube strips Axygen PCR-0208-AF-C
96-well PCR plate, skirted/semi-skirted such as IST scientific ISTSIST-601-096GCT
Self-adhesive aluminium foil LVL technologies AF100Plus
XSX 200 - 2D Biobanking Tubes LVL technologies 1C-X02-BL-CW-B-L

Stock reagents

Reagent Brand Catalogue number Storage
Deionized ultrapure water (ddH2O) Bionordika BN-51100 4 ºC
Absolute ethanol, EtOH 25 ºC
Elution Buffer (EB) Qiagen 19086 25 ºC
HighPrep™ PCR Clean-up System MAGBIO AC-60050 4 ºC
AmpliTaq Gold™ DNA Polymerase with Buffer II and MgCl2 Applied Biosystems N8080241 -20 ºC
dNTPs mix 10 mM each -20 ºC
IS7 primer for qPCR: ACACTCTTTCCCTACACGAC -20 ºC
IS8 primer for qPCR: GTGACTGGAGTTCAGACGTGT -20 ºC
SYBR® Green I Nucleic Acid Gel Stain - 10,000X concentrate in DMSO Invitrogen S7563 -20ºC
BioReagent Dimethyl sulfoxide, DMSO  Sigma Aldrich D2650-5X5ML 25 ºC
ROX Reference Dye Invitrogen 12223012 -20 ºC
  • Basic reagents for agarose gel electrophoresis (2% Agarose solution, GelRed® nucleic acid dye, PBS buffer, 1Kb DNA ladder)
  • P7 Index Primer: CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGT
  • P5 Index Primer: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCT

7.2 Protocol

1. Library QC qPCR

The efficiency of library preparation can vary significantly, especially when working with a diverse range of biological samples. This variation arises due to the presence of inhibitors, which differ greatly across taxa and sample types, and can substantially reduce the enzymatic efficiency of the reactions. Measuring the concentration or molarity of the library product proves ineffective, as it encompasses measurements of target DNA with attached adaptors, DNA lacking attached adaptors, loose adaptors, and adaptor dimers. Consequently, this approach fails to provide any meaningful information about the library’s effectiveness.

The most effective approach to evaluate library preparation quality involves conducting a qPCR assay utilizing PCR primers that hybridize with the adaptors linked to the DNA molecules during the library preparation step. Within the qPCR assay, only DNA molecules with adaptors attached to both ends and adaptor dimers undergo amplification. Unlike traditional PCR, qPCR offers a real-time overview of the amplification pattern, which proves invaluable for assessing the ideal library quantity, identifying inhibitor presence, and estimating the optimal number of PCR cycles required for the subsequent indexing PCR step in the pipeline.

Although the amplification pattern itself cannot distinguish between library amplification and adaptor-dimer amplification, the dissociation curve provided by qPCR platforms, along with the analysis of qPCR product via agarose gel electrophoresis, greatly assists in further evaluating library quality.

  1. Create the following PCR mastermix on a cooling block.
Reagent Stock concentration Mix concentration Volume per reaction
10x PCR Gold buffer 10X 1X 2.5 µl
MgCl2 Solution 25 mM 2.5 mM 2.5 µl
dNTPs Mix 10 mM each 0.08 mM each 0.2 µl
Forward (F) Primer 10 μM 0.4 μM 1.0 µl
Forward (R) Primer 10 μM 0.4 μM 1.0 µl
Sybr Green 1.0 µl
AmpliTaq GOLD DNA polymerase 5 U/µl 2.5 U 0.5 µl
ddH2O 14.3 µl
Total 23 µl
  1. Mix well and spin down mastermix.
  2. Aliquot 25 µl of the reaction to each well in the PCR plate/strip.
  3. Add 2 µl of 1:20 diluted library template to each well.
  4. Vortex and spin down the PCR mixture.
  5. Set up the qPCR program.
Step Time Repetition
95 ºC 12 min 1X
- - -
95 ºC 20 sec
60 ºC 30 sec 40 X
72 ºC 40 sec
- - -
Dissociation curve
  1. Run qPCR products on agarose gel 2% using 5 µl qPCR product + 2 µl dye solution. Settings: 130V, 350A, 35 minutes.

2. PCR amplification

  1. Create the PCR mastermix on a cooling block.
Reagent Stock concentration Mix concentration Volume per reaction
10x PCR Gold buffer 10X 1X 5.0 µl
MgCl2 Solution 25 mM 2.5 mM 5.0 µl
dNTPs Mix 10 mM each 0.08 mM each 0.4 µl
AmpliTaq GOLD DNA polymerase 5 U/µl 5 U 1.0 µl
ddH2O 26.6 µl
Total 38 µl
  1. Mix well and spin down mastermix.
  2. Aliquot 38 µl of the reaction to each well in the PCR plate/strip.
  3. Add 1 µl of each uniquely indexed primer to each well.
Reagent Stock concentration Mix concentration Volume per reaction
Forward (F) Primer 10 μM 0.2 μM 1.0 µl
Forward (R) Primer 10 μM 0.2 μM 1.0 µl
Total 2 µl
  1. Add 10 µl of DNA library product to each well.
  2. Vortex and spin down the PCR mixture.
  3. Set up the qPCR program with adjusted number of cycles per library, as determined by the qPCR screening.
Step Time Repetition
95 ºC 12 min 1X
- - -
95 ºC 20 sec
60 ºC 30 sec 7-19X
72 ºC 40 sec
- - -
72 ºC 5 min 1X
4 ºC inf. 1X

3. Magnetic bead-based purification

The final indexed library product needs to be purified in order to get rid of the enzymes and buffers employed in the PCR amplification.

  1. Equilibrate the beads to room temperature for 30 min.
  2. Ensure the beads are fully resuspended by vortexing.
  3. Transfer 60 µl (~ 1.2 times the volume of the library) of beads to each well containing the indexed library and mix thoroughly by pipetting.
  4. Incubate the mixture for 5 minutes at room temperature.
  5. Place PCR strips/PCR plate on a magnetic rack and wait until the supernatant is clear.
  6. Discard the supernatant.
  7. Add 200 µl of 80% EtOH to each well. Discard the supernatant.
  8. Repeat step 7 and ensure that all residual ethanol is removed.
  9. Dry the beads for a maximum of 5 minutes.
  10. Add 35 µl of EBT buffer to each well and quick-spin the PCR strips/PCR plate.
  11. Incubate the mixture 10 minutes at 37°C (outside the magnet).
  12. Quick-spin the PCR strips/PCR plate.
  13. Place the PCR strips/PCR plate on a magnetic rack and wait until the supernatant is clear.
  14. Aspirate (slowly to avoid bead transfer) and dispense the supernatant (DNA libraries) to a new PCR strips/PCR plate.
  15. Transfer the purified DNA to a 200 µl LVL tube.