• Automated NGS Nextera 16S metagenomic sequencing library preparation using the ASSIST PLUS pipetting robot

    A guide to automation of the Nextera 16S metagenomic library preparation

    The 16S metagenomic sequencing protocol allows targeted analysis of microbial communities by using the 16S ribosomal RNA (rRNA) gene – a trusted marker for phylogenetic classification – in combination with next generation sequencing (NGS) technology. This protocol ensures accurate amplicon sequencing, enabling the study of a diverse range of microbial populations.

    The workflow involves amplifying the variable regions 3 and 4 (V3 and V4) of the 16S rRNA gene with custom primers, incorporating Illumina sequencing adapters and dual indices for multiplexing. Using paired-end 300 bp reads, this approach delivers high quality data for taxonomic classification at the genus and species levels.

    This application guide provides programs for each of these steps for further optimization, helping you to automate 16S metagenomic sequencing library preparation on the ASSIST PLUS. 

  • Table of contents

    The 16S metagenomic sequencing protocol allows targeted analysis of microbial communities by using the 16S ribosomal RNA (rRNA) gene – a trusted marker for phylogenetic classification – in combination with next generation sequencing (NGS) technology. This protocol ensures accurate amplicon sequencing, enabling the study of a diverse range of microbial populations.

    The workflow involves amplifying the variable regions 3 and 4 (V3 and V4) of the 16S rRNA gene with custom primers, incorporating Illumina sequencing adapters and dual indices for multiplexing. Using paired-end 300 bp reads, this approach delivers high quality data for taxonomic classification at the genus and species levels.

    This application guide provides programs for each of these steps for further optimization, helping you to automate 16S metagenomic sequencing library preparation on the ASSIST PLUS. 

Key benefits

  • Optimized throughput: automates up to 24 samples per run, increasing efficiency for medium throughput laboratories.
  • Saves time and effort: simplifies complex workflows, freeing up valuable time for other tasks.
  • Walk-away convenience: 5 easy-to-adapt VIALAB template scripts enable hands-free operation, minimizing user intervention and manual handling.
  • Automated bead handling: standardized bead clean-up using the integrated MAG module enhances precision, minimizes errors and improves reproducibility.

Overview: How to perform automated 16S rRNA library preparation

The ASSIST PLUS pipetting robot streamlines 16S metagenomic sequencing library preparation while improving throughput, precision and reliability. Four preprogrammed VIALAB template scripts ensure efficient processing of the highlighted steps of 16S library preparation workflow (Figure 1) for up to 24 samples. For normalization and pooling, INTEGRA provides tested programs in other established app notes. These automated, walk-away protocols enhance user convenience and standardization for increased reproducibility and reduced hands-on time. Elevate your research with the ASSIST PLUS; achieve consistent and reliable results while saving valuable time.

The bead clean-up step is an integral part of 16S library prep and can easily be carried out using an INTEGRA MAG module. This magnetic separation device uses vertical magnet array movements to enable automated magnetic bead handling, avoiding unnecessary manual interventions.

Downloads: App note and programs

Overview of the 16S metagenomic sequencing library preparation workflow.
Figure 1: Overview of the 16S metagenomic sequencing library preparation workflow.

An overview of the key steps is presented in Figure 1. Each of these steps plays an essential role in the overall procedure and will be examined in detail in the following subsections.

First stage PCR

Protocol steps in VIALAB
Protocol steps in VIALAB
First stage PCR - workflow overview

This step employs PCR to amplify the DNA template using a primer that is specific to the region of 
interest and has overhang adapters attached.

Sample number: up to 24 
Estimated process time: 18 min

StepPhaseDescriptionManual intervention
1Initial volumesDefine initial reagent volumes in the labware on the ASSIST PLUS deckNo
2Master mixSet up the master mix
3Distribute master mixDistribute 22.5 µl of the master mix into each well of the plate
4-5Add DNA templateAdd 2.5 µl of DNA template per well and mix
6Seal plateManually seal the PCR plate and place it intoYes

 

ASSIST PLUS deck set-up

ASSIST PLUS deck set-up for the first stage PCR
Figure 2: The ASSIST PLUS deck set-up for first stage PCR. Position A: DNA samples used for library preparation in a Bio-Rad Hard-Shell® 96-Well PCR Plate (light blue). Position B: Rack for 1.5/2 ml microcentrifuge tubes with 3 tubes containing 2x concentrated KAPA HiFi HotStart ReadyMix in position A1 (green), forward and reverse primers in positions B1 and C1, respectively (pink). Position C: Target plate – empty PCR plate mounted on a COLDPLATE with a PCR plate adapter.

Reagents

ColorReagentVolume 
per sample (µl) 		Volume 
per 24 samples* (µl)		Deck
 position
 Microbial DNA template, 5 ng/µl50-A
 2x KAPA HiFi HotStart ReadyMix12.5330B
 Forward primer, 1 µM amplicon PCR5132B
 Reverse primer, 1 µM amplicon PCR 5132B

* including 10 % overhead

 

Materials and labware
Materials and labware

Instruments, modules and pipettes

Labware typeNameManufacturerPart no.
Pipette300 µl D-ONEINTEGRA4531
Tube rackRack for 2 ml microcentrifuge tubesINTEGRA4540
Cooling plateCOLDPLATEINTEGRA4950 (module)
4954 (adapter)

Consumables

Consumable typeNameManufacturerPart no.
PlateHard-Shell 96-Well PCR PlateBio-RadHSP9601
Tip12.5 µl sterile, filter GRIPTIPS®INTEGRA6455
Tip300 µl sterile, filter GRIPTIPS®INTEGRA6435
Protocol information
Protocol information

PCR cycling program

StepTemp. (°C)TimeCycles
Initial denaturation953 min1x
Denaturation9530 sec25x
Annealing5530 sec
Extension7230 sec
Final extension725 min1x
Hold4∞ (infinite)1x

First PCR clean-up

Protocol steps in VIALAB
Protocol steps in VIALAB
First PCR clean up - workflow overview

Following the addition of overhang adapters in first stage PCR, excess primers, nucleotides and enzymes must be removed to avoid interference in subsequent stages.

Sample number: up to 24 
Estimated process time: 46 min

StepPhaseDescriptionManual intervention
1Initial volumesDefine the initial reagent volumes in the labware on the ASSIST PLUS deckNo
2-8Add magnetic beadsAdd 20 µl of magnetic beads per sample
9-15Wash 1Wash using 125 µl of 80 % EtOH per sample
16-25Wash 2Wash again using 125 µl of 80 % EtOH per sample
26-33ElutionElute sample in 52.5 µl of elution buffer per sample

 

ASSIST PLUS deck set-up

ASSIST PLUS deck set-up for the first pcr clean up
Figure 3: The ASSIST PLUS deck set-up for first PCR clean-up. Position A: Reagents – INTEGRA deep well plate containing magnetic beads (green), wash buffer (80 % EtOH, pink) and elution buffer (lilac). Position B: Target plate – empty PCR plate for collection of cleaned PCR product. Position C: Source plate on MAG module – empty PCR plate for PCR clean-up with magnetic beads on a MAG module (light blue: DNA samples).

Reagents

ColorReagentVolume 
per sample (µl) 		Volume 
per 24 samples* (µl)		Deck
 position
 PCR amplification sample product25-C
 SPRI magnetic beads (e.g. MAGFLO NGS™)20528A
 80 % EtOH2506,600A
 Elution buffer501,320A

* including 10 % overhead

 

Materials and labware
Materials and labware

Instruments, modules and pipettes

Labware typeNameManufacturerPart no.
Pipette125 μl, 8 channel VOYAGER electronic pipetteINTEGRA4722
ModuleMAG with 96 well PCR adapter plateINTEGRA4900 (module)
4906 (adapter)

Consumables

Consumable typeNameManufacturerPart no.
Plate96 well deep well plate, polypropylene, sterile, 2.2 mlINTEGRA6353
PlateHard-Shell 96-Well PCR PlateBio-RadHSP9601
Tip125 µl sterile, filter GRIPTIPSINTEGRA6455

Second stage PCR

Protocol steps in VIALAB
Protocol steps in VIALAB
Second stage PCR - workflow overview

The second PCR cycle adds indexing barcodes and sequencing adapters, enabling sample multiplexing and compatibility with sequencing platforms.

Sample number: up to 24 
Estimated process time: 47 min

StepPhaseDescriptionManual intervention
1Initial volumesDefine the initial reagent volumes in the labware on the ASSIST PLUS deckNo
2-5Distribute primer 1Distribute 5 µl of N701-N703 Nextera primer into the plate
6-14Distribute primer 2Distribute 5 µl of S501-S508 Nextera primer into the plate
15Master mixSet up the master mix
16Distribute master mixDistribute 35 µl of the master mix into each well
17-18Add DNA templateAdd 5 µl of DNA template into each well and mix
19Seal plateManually seal the PCR plate and place it into a PCR cyclerYes 

 

ASSIST PLUS deck set-up

ASSIST PLUS deck set-up for the second stage PCR
Figure 4: The ASSIST PLUS deck set-up for second stage PCR. Position A: PCR plate containing DNA samples used for library preparation (green). Position B: Rack for 1.5/2 ml microcentrifuge tubes with 8+5 tubes containing Nextera index primer 2 (light blue), Nextera index primer 1 (pink), 2x KAPA HiFi HotStart ReadyMix (lilac) and PCR grade H2O (dark blue). Position C: Target plate – empty PCR plate on a COLDPLATE with a PCR plate adapter.

Reagents

ColorReagentVolume 
per sample (µl) 		Volume 
per 24 samples* (µl)		Deck
 position
 Nextera index primer 1 
N701 – 10 µM 
N702 – 10 µM 
N703 – 10 µM		544 eachB
 Nextera index primer 2 
S501 – 10 µM 
S502 – 10 µM 
S503 – 10 µM 
S504 – 10 µM 
S505 – 10 µM 
S506 – 10 µM 
S507 – 10 µM 
S508 – 10 µM		517 eachB
  2x KAPA HiFi HotStart ReadyMix 25660B
 PCR grade H2O 10264B
 Purified DNA template 5-A

* including 10 % overhead

 

Materials and labware
Materials and labware

Instruments, modules and pipettes

Labware typeNameManufacturerPart no.
Plate0.5-300 μl D-ONE INTEGRA4531
Plate Rack for 2 ml microcentrifuge tubes INTEGRA4540
Tip COLDPLATE INTEGRA4950 (module)
4954 (adapter)

Consumables

Consumable typeNameManufacturerPart no.
Plate Hard-Shell 96-Well PCR Plate Bio-Rad HSP9601
Tip12.5 µl sterile, filter GRIPTIPS INTEGRA 6455
Tip 300 µl sterile, filter GRIPTIPS INTEGRA6435
Protocol information
Protocol information

PCR cycling program

StepTemp. (°C)TimeCycles
Initial denaturation953 min1x
Denaturation9530 sec8x
Annealing5530 sec
Extension7230 sec
Final extension725 min1x
Hold4∞ (infinite)1x

Second PCR clean-up

Protocol steps in VIALAB
Protocol steps in VIALAB
Second stage PCR clean up - workflow overview

The second PCR clean-up step ensures excess primers, adapters and other contaminants are removed, resulting in a high quality, sequencing-ready 16S metagenomic library.

Sample number: up to 24 
Estimated process time: 48 min

StepPhaseDescriptionManual intervention
1Initial volumesDefine the initial reagent volumes in the labware on the ASSIST PLUS deckNo
2-8Add magnetic beadsAdd 56 µl of magnetic beads per sample
9-15Wash 1Wash using 125 µl of 80 % EtOH per sample
16-23Wash 2Wash again using 125 µl of 80 % EtOH per sample
24-29ElutionElute sample in 27.5 µl of elution buffer per sample

 

ASSIST PLUS deck set-up

ASSIST PLUS deck set-up for the second pcr clean up
Figure 5: The ASSIST PLUS deck set-up for the second PCR clean-up. Position A: Reagents – INTEGRA deep well plate containing magnetic beads (green), wash buffer (80 % EtOH, pink) and elution buffer (lilac). Position B: Target plate – empty PCR plate for collection of PCR clean-up product.  Position C: Source plate on MAG module – empty PCR plate for PCR clean-up with magnetic beads on a MAG module (light blue: DNA samples).

Reagents

ColorReagentVolume 
per sample (µl) 		Volume 
per 24 samples* (µl)		Deck
 position
 PCR amplification sample product of second stage PCR50-C
 SPRI magnetic beads (e.g. MAGFLO NGS)561,479A
 80 % EtOH2502,600A
 Elution buffer27.5726A

* including 10 % overhead

 

Materials and deck labware
Materials and deck labware

Instruments, modules and pipettes

Labware typeNameManufacturerPart no.
Pipette125 μl, 8 channel VOYAGER electronic pipetteINTEGRA4722
ModuleMAG with 96 well PCR adapter plateINTEGRA4900 (module)
4906 (adapter)

Consumables

Consumable typeNameManufacturerPart no.
Plate96 well deep well plate, polypropylene, sterile, 2.2 mlINTEGRA6353
PlateHard-Shell 96-Well PCR PlateBio-RadHSP9601
Tip125 µl sterile, filter GRIPTIPSINTEGRA6455

Conclusion

  • This comprehensive guide discusses free-of-charge VIALAB templates for 16S library preparation – from amplification to clean-up – providing a backbone for automated protocol development.
  • The detailed reagent and deck layout guidance provided here minimizes set-up errors and ensures reproducibility across multiple runs, supporting robust method standardization in diverse lab environments.
  • Combining the ASSIST PLUS with specialized modules, like the MAG and COLDPLATE, enables efficient magnetic bead separation and precise thermal control, supporting critical steps in PCR and purification.
  • The modular structure of the automated workflow on the ASSIST PLUS offers easy customization and scalability, making it adaptable to future protocol updates or changing sample input requirements.

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Instruments and accessories

ASSIST PLUS, Pipetting Robot D-ONE Single Channel Pipetting Module, 0.5-300 µl GRIPTIPS®, 12.5 µl, Sterile, Filter GRIPTIPS®, 125 µl, Sterile, Filter GRIPTIPS®, 300 µl, Sterile, Filter INTEGRA 96 square well, 2.2 ml deep well plate VOYAGER, 8 Channel, 125 µl, electronic pipette MAG module for magnetic separation COLDPLATE Rack for 1.5/2 ml microcentrifuge tubes Adapter for 96 well PCR plates (MAG/HEATMAG) MAGFLO™ NGS, 1 - 500 ml BioRad: Hard-Shell 96-well PCR plate, low profile, thin wall, skirted ILLUMINA: Nextera XT DNA Library Preparation Kit

ASSIST PLUS, Pipetting Robot

INTEGRA has developed the ASSIST PLUS pipetting robot to streamline routine pipetting tasks at an affordable price. Using INTEGRA electronic multichannel pipettes, the system:

  • automates pipetting tasks,
  • eliminates physical strain and
  • ensures superior reproducibility and
  • error free pipetting.

Part No. 4505

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ASSIST PLUS Pipetting Robot

D-ONE Single Channel Pipetting Module, 0.5-300 µl

The D-ONE single channel pipetting module offers hands-free transfers from individual tubes or wells on the ASSIST PLUS pipetting robot. This makes it the perfect add-on for labs that want to go beyond the possibilities of multichannel pipetting to access unlimited applications. It is designed to offer easy automation of normalization, hit picking and master mix preparation, freeing up your time while eliminating transcription errors.

Learn more about the D-ONE Single Channel Pipetting Module
D-ONE pipetting module 1ch 5-1,250ul

GRIPTIPS®, 12.5 µl, Sterile, Filter

GRIPTIPS® pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

Part No. 4415

Learn more about GRIPTIPS® pipette tips for Benchtop Pipetting Systems
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GRIPTIPS®, 125 µl, Sterile, Filter

GRIPTIPS® pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

Part No. 4425

Learn more about GRIPTIPS® pipette tips for Benchtop Pipetting Systems
Integra-Relaunch_Website-Output-Produktebilder-Product_Overview-GripTips.png

GRIPTIPS®, 300 µl, Sterile, Filter

GRIPTIPS® pipette tips perfectly match the multi-lobe tip fitting, snapping firmly on during loading. This guarantees a perfect seal on every tip, preventing them from loosening, leaking or completely falling off. All tips are precisely aligned horizontally, enabling accurate touch-offs, even when pipetting with 384 tips.

Part No. 3435

Learn more about GRIPTIPS® pipette tips for Handheld Pipettes
300 µl Pre-sterilized, Filter GripTips

INTEGRA 96 square well, 2.2 ml deep well plate

  • Maximum Volume of 2.4ml
  • Square well with pyramid bottom
  • Raised well rims for reliable closing with heat sealing and easy stacking
  • Good centrifugation stability up to 6,000 × g for faster protocols and improved sample quality
  • DNase/ RNase free, without slip agents, plasticizers or biocides

Part No. 6353

Learn more about our deep well plate
INTEGRA 96 square well, V bottom, polypropylene, sterile, 2.2 ml deep well plates

VOYAGER, 8 Channel, 125 µl, electronic pipette

VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

  • Single handed operation leaves the other hand free to handle labware.
  • On the fly access to up to 3 user-defined tip spacings.
  • These user defined tip spacings are saved. No need to memorize spacings of different labware formats.

Part No. 4722

See it work
VOYAGER pipettes allow the tip spacing to expand anywhere between 4.5 mm and 33 mm at the push of a button.

MAG module for magnetic separation

  • Adjustable magnet heights for bead collection, depending on the volume.
  • Exceptional magnet strength, ensuring minimal bead carryover and maximum yields.
  • Easy integration with the VIAFLO 96, MINI 96 or VIAFLO 384 pipetting platforms.
  • Full automation when combined with the ASSIST PLUS pipetting robot.

Part No. 4900

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HEATMAG module without adapter

COLDPLATE

  • Heating and cooling from 0 to 99°C
  • Can be fully automated on ASSIST PLUS pipetting robot or used stand-alone or on VIAFLO 96 and VIAFLO 384
  • Includes COLDPLATE, footprint adapter, labware pedestal (+50 mm), communication cable to ASSIST PLUS, communication cable to PC, cable clips
  • No labware adapter included

Part No. 4950

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coldplate for cooling and heating with flat bottom

Rack for 1.5/2 ml microcentrifuge tubes

Hold up to 48 tubes on removable carriers for easy loading and storage.

Part No. 4540

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Rack for 1.5/2 ml microcentrifuge tubes

Adapter for 96 well PCR plates (MAG/HEATMAG)

Part No. 4906

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Adapter and magnet for 384 well PCR plate

MAGFLO™ NGS, 1 - 500 ml

MAGFLO NGS magnetic beads provide a reliable solution for the purification of nucleic acid fragments with a consistent size distribution, which is crucial for NGS library preparation workflows. This includes both single- and double-sided size selection methods for DNA or RNA, as they are free of RNases. 

Part No. 7000/7002/7004

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MAGFLO ngs magnetic beads for ngs size selection 1ml 50ml 500ml

BioRad: Hard-Shell 96-well PCR plate, low profile, thin wall, skirted

Instrumentation compatible with low-profile PCR plates include Bio-Rad’s C1000 Touch and S1000 Thermal Cyclers and the CFX series real-time PCR systems. Use the PCR Plastics Consumables Selector Tool to determine individual plate compatibility with other instruments.

Source: Website BioRad

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ILLUMINA: Nextera XT DNA Library Preparation Kit

The Nextera XT DNA Library Preparation Kit offers a fast and easy workflow for preparing, quantifying, and normalizing libraries for sequencing small genomes, PCR amplicons, plasmids, microbial genomes, concatenated amplicons, and double-stranded cDNA from a low amount of input DNA (1 ng), making this kit amenable for use with precious samples available in limited quantity.


Part No. 15032354

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DownloadAutomated NGS Nextera 16S metagenomic sequencing library preparation using the ASSIST PLUS pipetting robot

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