Metagenomic sequencing with Oxford Nanopore involves several steps.

Here’s a high-level
overview:

1. DNA Extraction: The first step in any sequencing protocol is to extract DNA from your
sample. This can be done using a variety of methods, depending on the type of sample
and the desired quality and quantity of DNA.
2. Library Preparation: Once you have extracted DNA, you will need to prepare a library
for sequencing. This involves fragmenting the DNA into smaller pieces, attaching
adapters to the ends of the fragments, and amplifying the library if necessary.
3. Sequencing: The prepared library is then loaded onto a flow cell and sequenced using the
Oxford Nanopore device. The device uses nanopores to read the DNA sequences as they
pass through the pore.
4. Data Analysis: After sequencing, the data needs to be analyzed. This can involve base
calling (converting the raw signal into DNA sequence), quality control, alignment to a
reference genome (if available), and variant calling.

The Oxford Nanopore Protocol Builder is an interactive tool that enables you to generate your
own end-to-end protocol, with application-specific advice encompassing DNA extraction, library
prep, sequencing, and data analysis1.

Nanopore technology offers a variety of metagenomic approaches — from PCR-free whole-

genome sequencing through to amplicon-based 16S rRNA sequencing — that can be used in the
lab, field, or limited resource settings2.

Please note that this is a high-level overview and the exact protocol may vary depending on the
specific requirements of your experiment. Always refer to the manufacturer’s instructions and
guidelines when conducting your experiment12.

Nanopore sequencing offers several advantages for metagenomic research:

1. Unrestricted Read Lengths: Nanopore sequencing can generate reads of unrestricted

lengths, from short to ultra-long1. This enhances genome assembly and enables more
accurate analysis of known and novel microbes1. It also allows for precise differentiation
of closely related microbes1.
2. Real-Time Data Streaming: This technology enables immediate access to results, such
as species identification, relative abundance, and antimicrobial resistance (AMR)
analysis1.
3. Complete Genome and Plasmid Resolution: With nanopore sequencing, you can
resolve complete genomes and plasmids with reads of unrestricted length1.
4. Real-Time Identification: It allows for the identification of species, AMR, and virulence
factors in real time1.
 5. Portability: You can sequence at the sample source using portable devices like MinION
and Flongle1.
6. Streamlined Workflow: Nanopore sequencing can streamline your workflow with 10-
minute library preps1.
7. Detection of Base Modifications: It can detect base modifications and link plasmids to
hosts using epigenetic motifs1.
8. High-Contiguity Reference-Quality Genomes: Nanopore sequencing enables the
reconstruction of high-contiguity reference-quality genomes from complex metagenomes
directly using only Nanopore long reads2.
9. Improved Host Identification: The generated high-accuracy and high-contiguity
metagenome-assembled genomes (MAGs) improve the host identification of critical
mobile genetic elements, e.g., prophage2.

Please note that while Nanopore sequencing has difficulty fully characterizing long
homopolymer regions, introducing insertion/deletion errors, the continuous improvement of
sequencing accuracy, throughput, and theoretically unlimited read length empower much more
cost- and time-efficient genome reconstruction2.

2- library preparation:

Sure, here’s a more detailed step-by-step guide to library preparation for Next-Generation
Sequencing (NGS):

1. DNA Fragmentation: The first step in library preparation is DNA fragmentation. This
process breaks the DNA into smaller pieces. This can be done through various methods,
including sonication, enzymatic digestion, or nebulization.
2. End Repair: The fragmented DNA has overhangs that need to be repaired. The end
repair process converts the overhangs into blunt ends. This is done using an enzyme mix
that contains a DNA polymerase and a phosphatase.
3. A-Tailing: An ‘A’ base is added to the 3’ end of the blunt fragments. This is done using a
DNA polymerase that lacks proofreading activity. The ‘A’ base is necessary for the next
step, ligation.
4. Adapter Ligation: In this step, adapters with a ‘T’ overhang are ligated to the ‘A’ tailed
fragments. These adapters are necessary for the fragments to bind to the flow cell in the
sequencer. The ligation is done using a DNA ligase.
5. Size Selection: The fragments are then selected based on size. This is usually done using
gel or bead-based methods. The size selection step ensures that the library has a uniform
size distribution, which is important for efficient sequencing.
6. PCR Amplification: The DNA fragments are then amplified using PCR. This increases
the amount of DNA available for sequencing. The PCR step also adds index sequences to
the adapters, which are used for multiplexing during sequencing.
7. Quality Control: Finally, the quality and quantity of the library are assessed before
sequencing. This can be done using various methods, including qPCR, Bioanalyzer, or
TapeStation. The quality control step ensures that the library is suitable for sequencing.
Remember, each step is critical and any errors can lead to problems down the line. Therefore, it’s
important to use high-quality reagents and follow the protocols carefully. Happy sequencing! 😊

3- sequencing using the Oxford Nanopore device:

Sure, here’s a step-by-step guide to sequencing using the Oxford Nanopore device:

1. Library Loading: The prepared library is loaded onto a flow cell. The flow cell contains
an array of tiny holes, or nanopores, embedded in an electro-resistant membrane1.
2. Molecule Capture: Each nanopore corresponds to its own electrode connected to a
channel and sensor chip, which measures the electric current that flows through the
nanopore. When a molecule passes through a nanopore, the current is disrupted to
produce a characteristic ‘squiggle’1.
3. Sequencing: The squiggle is then decoded using basecalling algorithms to determine the
DNA or RNA sequence in real time1. Each base that passes through the nanopore can be
identified through the characteristic disruption it causes to the current1.
4. Real-Time Analysis: This makes nanopore sequencing unique, in that it is the only
sequencing technology that enables direct, real-time analysis of short to ultra-long
fragments of DNA/RNA, in fully scalable formats1.
5. Data Interpretation: The resulting data can be used to answer a range of biological
questions, providing information on pathogen identity, genetic disease risk, or how an
organism has evolved1.

Remember, each step is critical and any errors can lead to problems down the line. Therefore, it’s
important to use high-quality reagents and follow the protocols carefully.

For more detailed protocols, you can visit the Protocol Library in the Knowledge section of the
Oxford Nanopore Technologies website2. There, you can browse a growing directory of
Protocols, or plan your entire sequencing experiment using their new interactive Protocol builder
tool2.

4- DNA sequencing data analysis:

Absolutely, after sequencing, the data needs to be analyzed. Here’s a step-by-step guide to DNA
sequencing data analysis:

1. Quality Control (QC) of Raw Reads: The first step in data analysis is quality control.
This involves checking the raw sequencing reads for any errors or poor-quality sequences
that may have occurred during the sequencing process1.
2. Read Trimming: This step involves removing low-quality bases from the ends of the
reads, as well as any remaining adapter sequences1.
3. Alignment: The trimmed reads are then aligned to a reference genome. This helps to
identify the location of each read in the genome1.
4. Variant Calling: Once the reads have been aligned, the next step is to identify variants.
These are places in the genome where the sequenced DNA differs from the reference
genome1.
 5. Annotation: The identified variants are then annotated. This involves identifying the
genes that the variants are in and predicting the effect of the variants on the genes 1.
6. Interpretation: The final step is to interpret the results. This involves understanding
what the identified variants mean in the context of the research question1.

There are many tools and software available for each step of the data analysis process. The
choice of tool often depends on the specific requirements of the analysis1.

Remember, data analysis is a complex process that requires a good understanding of both the
biological question being asked and the statistical methods being used. Therefore, it’s important
to carefully plan your analysis and to thoroughly check your results at each step1.

For more detailed protocols, you can visit the Protocol Library in the Knowledge section of the
Oxford Nanopore Technologies website2. There, you can browse a growing directory of
Protocols, or plan your entire sequencing experiment using their new interactive Protocol builder
tool2.