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  • 1

    Product

  • 2

    Nanopore sequencing How it works

    Nanopore reader DNA or RNA passes through a nano-scale hole. The fluctuations in current during translocation are used to determine the DNA or RNA sequence.

    The nanopore processes the length of DNA or RNA presented to it. The user can control this through the library preparation protocol utilised. (e.g. >2 Mb DNA has been recorded.)

    An electrically resistant membrane means all current must pass through the nanopore, ensuring a clean signal.

    An enzyme motor controls the translocation of the DNA or RNA strand through the nanopore. Once the DNA or RNA has passed through, the motor protein detaches and the nanopore is ready to accept the next fragment.

    The nanopore signal, captured by the ASIC in the device, is characteristic of the sequence of the DNA or RNA fragment. Algorithms are used to convert the signal into basecalls.

  • 3

    Sequencing with 1D and 1D2 reads

    Library prep Whether using 1D or 1D2, library preparation results in the addition of a sequencing adapter at each end of the fragment. Both the template and complement strands carry the motor protein which means both strands are able to translocate the nanopore.

    Y-adapter Y-adapter

    Library DNA

    Template... Next molecule…(Exit)...Template... Template... ...Template... ...Complement(Exit)

    Translocation – 1D2 1D2 library preparation deploys special adapters that increase the probability that the complement strand will immediately follow the template strand. This method of sequencing when used with 1D2 analysis produces a higher accuracy read.

    Translocation – 1D The template and the complement strands are sequenced as individual strands.

    nanoporetech.com

  • SequencePrepare Analyse

    4

    DNA library preparation For maximum throughput For minimal preparation time

    Ligation Sequencing Kit Rapid Sequencing Kit with transposase

    • The transposase simultaneously cleaves template molecules and attaches tags to the cleaved ends

    • Rapid sequencing adapters are added to the tagged ends • Fragment lengths are a result of the random cleavage

    • DNA ends are repaired and dA-tailed • Sequencing adapters are ligated onto the prepared ends • Fragment lengths can be controlled by fragmentation or size selection

    60 min

    High molecular weight gDNA

    Optional fragmentation or size selection

    End-prep

    Tether attachment

    Loading

    1D PCR-free gDNA

    Ligation of sequencing adapters T

    p A

    A p

    5 min

    5 min

    Attachment of sequencing adapters

    Loading

    Transposome complex

    Cleavage and addition of transposase adapters

    gDNA

    LSK108

    RAD004

    60 min

    High molecular weight gDNA

    Optional fragmentation or size selection

    End-prep and nick repair

    Loading

    1D PCR-free gDNA

    Ligation of sequencing adapters T

    p A

    A p

    LSK109

    60 min

    High molecular weight gDNA

    Optional fragmentation or size selection

    End-prep

    Tether attachment

    Loading

    1D PCR-free gDNA

    Ligation of sequencing adapters T

    p A

    A p

    5 min

    5 min

    Attachment of sequencing adapters

    Loading

    Transposome complex

    Cleavage and addition of transposase adapters

    gDNA

    LSK108

    RAD004

    60 min

    High molecular weight gDNA

    Optional fragmentation or size selection

    End-prep and nick repair

    Loading

    1D PCR-free gDNA

    Ligation of sequencing adapters T

    p A

    A p

    LSK109

    60 min

    High molecular weight gDNA

    Optional fragmentation or size selection

    End-prep

    Tether attachment

    Loading

    1D PCR-free gDNA

    Ligation of sequencing adapters T

    p A

    A p

    5 min

    5 min

    Attachment of sequencing adapters

    Loading

    Transposome complex

    Cleavage and addition of transposase adapters

    gDNA

    LSK108

    RAD004

    60 min

    High molecular weight gDNA

    Optional fragmentation or size selection

    End-prep and nick repair

    Loading

    1D PCR-free gDNA

    Ligation of sequencing adapters T

    p A

    A p

    LSK109

  • SequencePrepare Analyse

    5

    Which DNA kit? These library preparation kits are all PCR-free, allowing sequencing without PCR bias

    Use for… Highest throughput Rapid and simple prep Highest raw read accuracy

    Prep time 60 mins 10 mins 80 mins

    Input amount 1000 ng dsDNA 400 ng HMW gDNA (>30 kb) 1000 ng dsDNA

    Fragmentation Optional Transposase based Optional

    Read length Equal to fragment length Random distribution, dependent on input fragment length Equal to fragment length

    Ligation 1D2 (SQK-LSK308)

    Rapid (SQK-RAD004)

    Ligation 1D (SQK-LSK109)

    Also available:

    • Optimised PCR-based kits for low input amounts • Application-specific library preparation kits (e.g. 16S sequencing) • Barcoding kits for cost-effective analysis of multiple samples

    View all kits and full specifications at store.nanoporetech.com

  • SequencePrepare Analyse

    6

    • Optional reverse transcription step improves throughput – cDNA strand is not sequenced

    • Sequencing adapters attached to prepared ends • Read length reflects length of molecules in sample

    • cDNA is synthesised using reverse transcription and strand-switching method, and then is amplified with PCR

    • Strand-switching before PCR enriches for full-length transcripts • Sequencing adapters are attached to the amplified cDNA

    100 min

    Loading

    Reverse transcription

    AAAAAAAAAAAAAAAAA

    Full-length RNA

    Primer annealing and ligation

    Attachment of 1D sequencing adapter

    and dual tethers15 min

    AAAAAAAAAAAAAAAAAA TTTTTT

    AAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTTT

    +

    AAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTTT

    TTTTTT

    F R

    Loading

    Reverse transcription and strand-switching

    Primer annealing TTTTTT

    Full-length RNA

    Attachment of rapid 1D sequencing adapters

    PCR with rapid attachment primers

    40 min

    30 min

    55 min

    AAAAAAAAAAAAAAAAA

    TTTTTTTTTT AAAAAAAAAA AA

    AA AA A

    TTTTTTTTTTCCC AAAAAAAAAAAA

    AA AA A

    AAAAAAAAAA TTTTTTTTTT

    GGG CCC

    AAAAAAAAAA TTTTTTTTTT

    GGG CCC

    GGG

    100 min

    Loading

    Reverse transcription

    AAAAAAAAAAAAAAAAA

    Full-length RNA

    Primer annealing and ligation

    Attachment of 1D sequencing adapter

    and dual tethers15 min

    AAAAAAAAAAAAAAAAAA TTTTTT

    AAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTTT

    +

    AAAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTTT

    TTTTTT

    F R

    Loading

    Reverse transcription and strand-switching

    Primer annealing TTTTTT

    Full-length RNA

    Attachment of rapid 1D sequencing adapters

    PCR with rapid attachment primers

    40 min

    30 min

    55 min

    AAAAAAAAAAAAAAAAA

    TTTTTTTTTT AAAAAAAAAA AA

    AA AA A

    TTTTTTTTTTCCC AAAAAAAAAAAA

    AA AA A

    AAAAAAAAAA TTTTTTTTTT

    GGG CCC

    AAAAAAAAAA TTTTTTTTTT

    GGG CCC

    GGG

    RNA library preparation For sequencing the RNA molecule directly For full-length transcript analysis with high throughput

    Direct RNA Sequencing Kit cDNA-PCR Sequencing Kit

  • SequencePrepare Analyse

    7

    Barcoding options for the cDNA kits are available, and are in development for the Direct RNA Sequencing Kit.

    Use for… Sequence RNA molecules directly and preserve base

    modifications Full-length transcripts with

    high throughput Full-length transcripts

    without PCR bias

    Prep time 115 mins 125 mins 210 mins

    Input recommendation 500 ng RNA (poly A+) 50 ng RNA (poly A+) 250 ng RNA (poly A+)

    Read length Equal to RNA length Enriched for full-length cDNA Enriched for full-length cDNA

    PCR required No Yes No

    Reverse transcription Optional Yes Yes

    Direct cDNA (SQK-DCS108)

    cDNA-PCR (SQK-PCS108)

    Direct RNA (SQK-RNA001)

    Which RNA kit?

    For full specifications visit store.nanoporetech.com

  • SequencePrepare Analyse

    8

    Maximising flow cell usage

    Barcoding

    Barcoding kits allow users to multiplex samples to generate maximum data from a single flow cell, to separate the reads from sequential library loadings and to lower the cost per sample.

    • Native Barcoding Kit for a PCR-free approach • PCR Barcoding Kits for up to 96 samples • Barcode libraries of gDNA, amplicon or cDNA either with

    a dedicated barcoding kit or a barcoding expansion pack

    Washing

    The wash kit allows re-use of flow cells after short sequencing runs, meaning multiple libraries can be run sequentially.

    Barcode multiple samples

    Separate and analyse

    Pool and sequence

    Visit store.nanoporetech.com for more details

  • SequencePrepare Analyse

    9

    VolTRAXTM Automated library preparation solution for nanopore sequencing

    Consumable car

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