Amplicon Library Preparation for Sequencing

SOP-L704 Revision 0.1
Author

Marc Los Huertos

EA Program, Pomona College

Published

2026-06-26

1 Purpose

This SOP describes the preparation of amplicon sequencing libraries for Illumina platforms, following a two-step PCR approach. The first PCR amplifies the target marker gene (e.g., 16S rRNA for bacteria, ITS for fungi) with gene-specific primers tailed with Illumina adapter overhangs. The second PCR attaches unique dual-index barcodes and the full Illumina adapter sequences, enabling multiplexed sequencing of many samples per run. This protocol is based on the Illumina 16S Metagenomic Sequencing Library Preparation guide and the Earth Microbiome Project (EMP) standards (Caporaso et al., 2012; Illumina, Inc., 2013; Thompson et al., 2017).

2 Scope

  • Applies to: Preparation of amplicon libraries targeting 16S rRNA (V4 or V3–V4 region), ITS (ITS1 or ITS2 region), 18S rRNA, or other marker genes from environmental DNA extracts for paired-end sequencing on Illumina MiSeq or NextSeq platforms
  • Does not apply to: Shotgun metagenomic library preparation; long- read amplicon sequencing (PacBio, Nanopore); ddPCR (see SOP-L701); eDNA metabarcoding using non-Illumina workflows
  • Location: Molecular biology bench, SGM 133

3 Responsibilities

Role Responsibility
Lab PI Approve target region, primer choice, and sequencing facility
Trained Personnel Prepare libraries per this SOP; submit to sequencing facility
New Students Complete training before independent use

4 Definitions

  • Adapter: A short double-stranded DNA sequence ligated to both ends of a library fragment; contains the sequencing primer binding site, an index sequence, and the flow cell binding sequence required for Illumina cluster generation.
  • Amplicon library: A sequencing library prepared by PCR amplification of a target region (e.g., 16S rRNA gene V4 region or ITS2) using primers that incorporate Illumina adapter sequences; represents the diversity of that locus in a community sample.
  • Index (barcode): A unique 6–12 bp sequence incorporated into the adapter of each sample’s library; allows multiple samples to be pooled and sequenced together (multiplexed) and computationally separated (demultiplexed) after sequencing.
  • Library normalization: Adjustment of each sample’s library to the same molar concentration before pooling; ensures approximately equal sequencing depth across samples; performed by quantification (Qubit or qPCR) followed by dilution.
  • Library pooling: Combining equimolar amounts of individually indexed libraries from multiple samples into a single tube for multiplexed sequencing; the total library pool is loaded onto the sequencer.
  • Paired-end sequencing: A sequencing strategy in which both ends of a DNA insert are sequenced, producing two reads (R1 and R2) per cluster; paired reads overlap for amplicons shorter than twice the read length, enabling error correction by merging.
  • PCR inhibitor: A substance carried over from DNA extraction that suppresses amplification efficiency (e.g., humic acids, tannins, residual ethanol); detected by inhibition testing and remedied by further purification or dilution of the template.
  • Size selection: A step in library preparation that removes off-target fragments (primer dimers, non-specific products) using bead-based cleanup (e.g., AMPure XP beads) or gel excision; improves sequencing quality and reduces wasted reads.

5 Safety and Hazards

WarningHazards
  • Chemical: AMPure XP beads (or equivalent SPRI beads) contain polyethylene glycol (PEG) and carboxylated magnetic particles; low toxicity but handle with gloves. Molecular-grade ethanol is flammable.
  • Biological: Environmental DNA extracts may contain pathogen-derived DNA. Handle with standard precautions.

5.1 Required PPE

5.2 Emergency Procedures

Refer to SOP-L003 Hazardous Materials Handling.

6 Equipment and Materials

6.1 Equipment

Item Location Notes
Thermal cycler Mol bio bench For both PCR1 and PCR2
Magnetic stand (for 96-well plates or strip tubes) Mol bio bench For SPRI bead cleanup
Qubit 4 fluorometer Instrument bench For library quantification
NanoDrop One Instrument bench Optional purity check
Gel electrophoresis system Mol bio bench For amplicon verification; see SOP-L703
Multichannel pipette (optional) Mol bio bench For high-throughput plate work
Plate vortex mixer Mol bio bench For mixing bead suspensions
Microcentrifuge and plate spinner Mol bio bench For brief spin-downs

6.2 Reagents and Consumables

Item Notes
High-fidelity DNA polymerase e.g., KAPA HiFi HotStart (Roche), Q5 (NEB), or Phusion (Thermo Fisher); high-fidelity polymerase is required to minimize PCR-introduced errors (Schirmer et al., 2015)
Gene-specific primers with Illumina overhangs (PCR1) See Section 6.1
Nextera XT Index Kit v2 (or equivalent dual-index primers) For PCR2 barcoding
AMPure XP beads (Beckman Coulter) or equivalent SPRI beads For size selection and cleanup
Molecular-grade ethanol (80%, freshly prepared) For bead washes
Nuclease-free water For elution and dilutions
Qubit dsDNA HS Assay Kit For library quantification
Agarose and gel supplies For amplicon verification
PCR plates or strip tubes (0.2 mL) Low-bind preferred
Plate sealing film or strip caps
Filter pipette tips Barrier tips mandatory

7 Procedure

7.1 Primer Design (PCR1: Gene-Specific Amplification)

Primers for the first PCR consist of the gene-specific sequence plus the Illumina adapter overhang. The overhang sequences are:

  • Forward overhang: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[gene-specific forward]-3'
  • Reverse overhang: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[gene-specific reverse]-3'

Commonly used gene-specific primers:

Target Primers Region Amplicon Reference
16S bacteria/archaea 515F / 806R V4 ~290 bp Caporaso et al. (2012); Parada et al. (2016)
16S bacteria 341F / 785R V3–V4 ~460 bp Klindworth et al. (2013)
ITS fungi ITS1f / ITS2 ITS1 ~250–400 bp Smith & Peay (2014); White et al. (1990)
ITS fungi fITS7 / ITS4 ITS2 ~250–400 bp Ihrmark et al. (2012)
18S eukaryotes Euk1391f / EukBr V9 ~130 bp Amaral-Zettler et al. (2009)
Note

The EMP standard primers for 16S V4 are 515F (Parada) / 806R (Apprill), which include degeneracies for improved archaeal coverage (Apprill et al., 2015; Parada et al., 2016). Use these unless the project specifically requires a different region.

7.2 PCR1: Target Amplification

  1. Prepare the master mix on ice. Use a high-fidelity polymerase.
Component Volume per reaction Final concentration
2x KAPA HiFi HotStart ReadyMix 12.5 uL 1x
Forward primer with overhang (10 uM) 1.0 uL 400 nM
Reverse primer with overhang (10 uM) 1.0 uL 400 nM
Nuclease-free water 8.5 uL –
Template DNA (1–10 ng) 2.0 uL Variable
Total 25.0 uL
  1. Include controls: positive control, NTC, and extraction blank.
  2. Cycling parameters (adjust annealing temperature per primer set):
Step Temperature Time Cycles
Initial denaturation 95 C 3 min 1
Denaturation 95 C 30 s 25–28
Annealing 55 C 30 s 25–28
Extension 72 C 30 s 25–28
Final extension 72 C 5 min 1
Hold 4 C Hold –
Important

Limit PCR1 to 25–28 cycles to minimize chimera formation and PCR bias (Schirmer et al., 2015). More cycles increase the proportion of artifacts in the sequencing data.

  1. Verify amplification by running 2 uL on a 1.5% agarose gel per SOP-L703.

7.3 PCR1 Cleanup (SPRI Beads)

  1. Allow AMPure XP beads to reach room temperature (approximately 30 minutes). Vortex well before use.
  2. Add 20 uL (0.8x ratio) of beads to each 25 uL PCR product. Mix by pipetting 10 times.
  3. Incubate at room temperature for 5 minutes.
  4. Place on the magnetic stand for 2 minutes until the solution clears.
  5. Aspirate and discard the supernatant.
  6. Wash twice with 200 uL of freshly prepared 80% ethanol. Do not disturb the bead pellet. Allow 30 seconds per wash.
  7. Remove all ethanol and air-dry the beads for 3–5 minutes. Do not over-dry (beads will crack and yield drops).
  8. Remove from the magnetic stand. Elute in 27.5 uL of nuclease-free water. Pipette to mix.
  9. Return to the magnetic stand. Transfer 25 uL of the clear eluate to a new tube.

7.4 PCR2: Index PCR (Barcoding)

  1. Assign a unique dual-index combination (i7 + i5) to each sample. Record the plate map in the lab notebook and in the sample sheet.
  2. Set up the index PCR:
Component Volume per reaction
2x KAPA HiFi HotStart ReadyMix 12.5 uL
Nextera XT Index Primer 1 (i7) 2.5 uL
Nextera XT Index Primer 2 (i5) 2.5 uL
Cleaned PCR1 product 2.5 uL
Nuclease-free water 5.0 uL
Total 25.0 uL
  1. Cycling parameters:
Step Temperature Time Cycles
Initial denaturation 95 C 3 min 1
Denaturation 95 C 30 s 8
Annealing 55 C 30 s 8
Extension 72 C 30 s 8
Final extension 72 C 5 min 1
Hold 4 C Hold –
  1. Clean the indexed PCR product with AMPure XP beads (0.8x ratio) as in Section 6.3. Elute in 27.5 uL.

7.5 Library Quantification and QC

  1. Quantify each library using the Qubit dsDNA HS Assay (SOP-L700, Section 6.4).
  2. Verify library size by running 1 uL on a 1.5% agarose gel. The expected size is the amplicon plus approximately 120 bp of adapter and index sequence.
  3. Libraries with concentrations below 1 nM may not sequence well. Re-amplify or exclude them.

7.6 Library Pooling and Submission

  1. Normalize each library to an equimolar concentration (typically 4 nM) by diluting with nuclease-free water.
  2. Pool equal volumes of all normalized libraries into a single tube.
  3. Quantify the pooled library by Qubit.
  4. Prepare the sample sheet (Illumina Experiment Manager format or the sequencing facility’s template) listing every sample with its index combination.
  5. Submit the pooled library and sample sheet to the sequencing facility per their submission guidelines.
  6. Record the submission date, facility, run type (e.g., MiSeq 2x250 or 2x300), and expected turnaround in the lab notebook.

8 Quality Control

  • Every library prep run must include: positive control (mock community, if available), NTC, and extraction blank carried through both PCR1 and PCR2
  • NTC and extraction blanks must show no visible band on the verification gel and no detectable concentration on the Qubit. If blanks are positive, the entire batch is suspect (Salter et al., 2014).
  • Verify amplicon size on gel after PCR1; verify library size (amplicon
    • adapters) on gel after PCR2
  • Record the number of PCR1 cycles and the index assignments for every sample; these are required for data analysis and troubleshooting

9 Waste Disposal

Waste Type Container Disposal Method
Used SPRI beads Standard trash Non-hazardous
Ethanol wash waste Chemical waste or evaporate in fume hood Small volumes can evaporate
Used PCR tubes, plates, tips Standard trash Non-hazardous
Gel waste Per SOP-L703 Depends on stain used

10 References

Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W., & Huse, S. M. (2009). A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PLOS ONE, 4(7), e6372. https://doi.org/10.1371/journal.pone.0006372
Apprill, A., McNally, S., Parsons, R., & Weber, L. (2015). Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquatic Microbial Ecology, 75(2), 129–137. https://doi.org/10.3354/ame01753
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., Owens, S. M., Betley, J., Fraser, L., Bauer, M., Gormley, N., Gilbert, J. A., Smith, G., & Knight, R. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME Journal, 6(8), 1621–1624. https://doi.org/10.1038/ismej.2012.8
Ihrmark, K., B"odeker, I. T. M., Cruz-Martinez, K., Friberg, H., Kubartova, A., Schenck, J., Strid, Y., Stenlid, J., Brandstr"om-Durling, M., Clemmensen, K. E., & Lindahl, B. D. (2012). New primers to amplify the fungal ITS2 region – evaluation by 454-sequencing of artificial and natural communities. FEMS Microbiology Ecology, 82(3), 666–677. https://doi.org/10.1111/j.1574-6941.2012.01437.x
Illumina, Inc. (2013). 16S metagenomic sequencing library preparation. Illumina, Inc. https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf
Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., & Gl"ockner, F. O. (2013). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research, 41(1), e1. https://doi.org/10.1093/nar/gks808
Parada, A. E., Needham, D. M., & Fuhrman, J. A. (2016). Every base matters: Assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environmental Microbiology, 18(5), 1403–1414. https://doi.org/10.1111/1462-2920.13023
Salter, S. J., Cox, M. J., Sherrill-Mix, S., Turner, R. M., Sherrill-Mix, S., Sherrill-Mix, S., & Walker, A. W. (2014). Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biology, 12, 87. https://doi.org/10.1186/s12915-014-0087-z
Schirmer, M., Ijaz, U. Z., D’Amore, R., Hall, N., Sloan, W. T., & Quince, C. (2015). Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Research, 43(6), e37. https://doi.org/10.1093/nar/gku1341
Smith, D. P., & Peay, K. G. (2014). Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLOS ONE, 9(2), e90234. https://doi.org/10.1371/journal.pone.0090234
Thompson, L. R., Sanders, J. G., McDonald, D., Amir, A., Ladau, J., Locey, K. J., Prill, R. J., Tripathi, A., Gibbons, S. M., Ackermann, G., Navas-Molina, J. A., Janssen, S., Kopylova, E., V’azquez-Baeza, Y., Gonz’alez, A., Morton, J. T., Mirarab, S., Xu, Z. Z., Jiang, L., … The Earth Microbiome Project Consortium. (2017). A communal catalogue reveals Earth’s multiscale microbial diversity. Nature, 551(7681), 457–463. https://doi.org/10.1038/nature24621
White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315–322). Academic Press. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

12 Training Record

Before preparing amplicon libraries independently, personnel must:

  1. Read this SOP, SOP-L703, and SOP-L700 in full
  2. Demonstrate SPRI bead cleanup with correct bead ratios and wash steps
  3. Prepare a supervised library from positive control DNA through pooling, with clean controls and correct library size on gel
  4. Complete the sequencing facility’s sample submission training
  5. Be signed off by the PI or lab manager