The ElysION automated Microbial Amplicon Barcoding Kit 24 V14 (SQK-MAB114.24) workflow is optimised for simplicity and speed to automatically prepare and sequence between 1 and 24 amplicon samples. Due to the presence of both highly conserved (adequate for universal primers and phylogenetic signal) and highly variant regions (different across species), the 16S rRNA gene can be used for bacterial profiling and ITS for fungal profiling from diverse sample types.

The Microbial Amplicon Barcoding Kit 24 V14 includes 16S and ITS primers designed to amplify commonly used regions for microbial identification. This targeted approach focuses on sequencing informative regions, helping you to characterise the composition of your samples without sequencing unnecessary parts of the genome, making experiments faster and more cost effective. This kit also contains 24 unique barcodes which allows you to pool up to 24 samples into one sequencing experiment, while maintaining full-length amplicon information.

After sequencing, the ElysION can perform downstream analysis using the EPI2ME 16S workflow to classify 16S and ITS amplicons from your samples. If you wish for the analysis to be automatically performed after sequencing, select either the 16S or ITS assay.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

• Ensure your ElysION device is installed with the required hardware and software package for this workflow.
• Ensure you have your sequencing kit, the correct equipment and third-party reagents.
• Check your flow cell during the ElysION run setup to ensure it has sufficient pores for a good sequencing run.

Automated library preparation, sequencing, and analysis

The Table below is an overview of the steps automated by the ElysION device.

Library preparation step	Process
16S or ITS PCR amplification	Amplify the 16S or ITS gene using primers supplied in the kit.
Amplicon barcoding	Attach barcodes for up to 24 amplicon samples.
Barcode inactivation, sample pooling and bead clean-up	Inactivate the barcoding reaction, pool the barcoded samples and perform a sample clean-up.
Priming and loading the flow cell	Prime the flow cell and load the prepared library for sequencing.
Sequencing	The sequencing run uses the MinKNOW software, which will collect raw data to basecall and demultiplex the barcoded reads.
Data analysis	Once sequencing is complete, there is a wf-16S workflow available to analyse your data through EPI2ME.
Flow cell wash or flush	Wash the flow cell using the Flow Cell Wash Kit (EXP-WSH004) for reuse or flush the flow cell for return to Oxford Nanopore for recycling.

Input DNA

How to QC your input DNA

It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, read the Input DNA/RNA QC protocol.

Chemical contaminants

Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

The number of pores in your flow cell will be checked by the ElysION device at the start of your assay. MinION/GridION Flow Cells should be checked within 12 weeks of purchasing.

Oxford Nanopore will replace any unused flow cell with fewer than the number of pores listed in the Table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, follow the instructions on the ElysION on-screen display.

Flow cell	Minimum number of active pores covered by warranty
MinION/GridION Flow Cell	800

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

名称	缩写	管盖颜色	管数	每管溶液体积 (μl)
快速测序文库接头	RA	绿色	1	15
16S 引物	16S	白色	1	800
ITS 引物	ITS	白色	1	800
短片段缓冲液	SFB	透明管盖, 白色标签	2	1800
EDTA	EDTA	蓝色	1	700
接头缓冲液	ADB	透明	1	100
AMPure XP 磁珠	AXP	棕色	2	1200
洗脱缓冲液	EB	黑色	1	500
测序缓冲液	SB	红色	1	700
文库颗粒	LIB	粉色	1	600
文库溶液	LIS	白色管盖，粉色标签	1	600
测序芯片冲洗液	FCF	透明管盖，浅蓝色标签	1	8000
冲洗系绳 UL	FTU	紫色	1	65
扩增子条形码 01-24	AB24	-	两板，每板三套条形码组合	每孔 5 μl

本产品包含由贝克曼库尔特公司（Beckman Coulter, Inc）生产的 AMPure XP 试剂，并可与试剂盒一起于 -20°C 下储存（试剂稳定性将不受损害）。

The sample sheet assigns a barcode to each sample, allowing you to pick a range from the 96-well plate, and for a sample ID to be tracked from input to results.

For more information on setting up a sample sheet, refer to the ElysION MinION user guide.

Ensure you have all the correct hardware components, software packages, and workflows installed to carry out this method.

For more information on the device and the processes, refer to the ElysION MinION user guide.

Pause for QC post PCR

Pause for QC pre Flow Cell Loading

Interleaving run setup

For commonly encountered issues, refer to the ElysION MinION user guide.

For in-depth troubleshooting, refer to the ElysION Operating manual provided with your device.

For additional customer support, contact the Oxford Nanopore support channel (support@nanoporetech.com).