Protocol from library to sequences

--- tags: molecular biology (tutorial), ebook, sequencing --- # Protocol from library to sequences ##### *Version v1.0: 2023-06-08* ### Overview and Objective The small MinION sequencing device uses “nanopore sequencing”, a process by which changes in the ionic current measured when a single-stranded DNA fragment is funneled through a biological pore in the device’s membrane can be converted into a nucleotide sequence. By sequencing using MinION, you can generate data in real time in a few hours (for smaller runs) or, in the case of high-throughput multiplexing (hundreds of samples), up to 72 hours. This protocol will sequence libraries with a MinION instrument. It is based on the protocols and resources available on Nanopore’s Resource Center and other literature sources. This protocol does not cover DNA extraction (there are several methods and commercial options that can be optimized for the group of interest covered by this ebook) or PCR (see *Protocol X*). Other protocols can be used in conjunct with different steps of this protocol (e.g., protocol to make home-made beads, [Qubit quantification](https://hackmd.io/20P-iQi6QH6B5BqfO6_Kww), etc.). --- ### *Materials* #### ONT sequencing preparation 1. ONT Sequencing Kit (ONT, cat. no. SQK-LSK109 or SQK-LSK110) 2. ONT Library Loading Kit 3. Nuclease-free water (e.g., Thermo Fisher, cat. no. AM9937) 4. (Optional) Flow Cell Wash Kit (ONT, cat. no. EXP-WSH004) – if using regular flow-cell instead of Flongle. 5. Flow Cell or Flonge :::warning :warning: Make sure to check if R9 or R10 flowcell is being used! ::: #### Standard equipment and consumables 1. Lab coats, gloves, and other PPE 2. P1000, P200, P10 pipette and filtered pipette tips 3. 1.5 ml Eppendorf DNA LoBind tubes (e.g., Eppendorf, cat. no. 022431021) 4. 0.2 ml strip thin-walled PCR tubes (e.g., Thermo Fisher Scientific, cat. no. AB0451) 5. Benchtop microcentrifuge (e.g., Thermo Fisher Scientific mySPIN 6, cat. no. 75004061) 6. Vortex or mixer or similar 7. Ice or frozen cool packs 8. (Optional) Qubit Fluorometer (Thermo Fisher Scientific) or (optional) TapeStation 2200 (Agilent) #### Sequencing preparation (using the SQK–LSK110 sequencing kit) :::info :bulb: Get some ice or frozen cool packs ready prior to start protocol ::: 1. Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII), Flush Tether (FLT) and one tube ofFlush Buffer (FB) at RT 2. Place the tubes on ice as soon as thawing is complete. 3. Mix the SBII and FB tubes by pipetting the liquid up and down, spin down and return to ice. 4. Spin down the FLT tube, mix by pipetting and return to ice. 5. Prepare final library for sequencing following the steps below: Follow **option A** if using a standard MinION flow cell, or **option B** if using a Flongle. :::warning :warning: ONT recommends loading **5–50 fmol** of the final prepared library onto R9.4.1 flow cells. For R10.3 flow cells, recommendation is to load **25–75 fmol**. If using the Flongle, ONT recommends loading **3–20 fmol** instead. **Critical:** Loading more than the maximum fmol recommended can have detrimental effects on throughput. Dilute the library in Elution Buffer if required. To calculate the right fmol, you can use several options online, like the [Promega online tool](https://promega.com/resources/tools/biomath). RECOMMENDATION: We recommend checking [ONT’s current protocol for updates and changes](https://community.nanoporetech.com/protocols). Also worth checking [ONT’s YouTube channel](https://www.youtube.com/channel/UC5yMlYjHSgFfZ37LYq-dzig) for updated introductory videos. ::: **A. Preparing and loading the Standard MinION flow cell** (i) Priming and loading: open the lid of the nanopore sequencing device, and slide the flow cell’s priming port cover clockwise so that the priming port is visible. (ii) Set a P1000 pipette to 200 μl. (iii) Insert the tip into the priming port. After opening the priming port, check for a small bubble under the cover. Draw back a small volume to remove bubble (20–30 μl). To do so, turn the pipette wheel until the dial shows 220–230 μl, or until you can see a small volume of buffer entering the pipette tip. Discard the liquid afterwards. Critical: Proceed with a lot of care when drawing back buffer from the flow cell. The array of pores must be covered by buffer at all times. :::warning :warning: Removing >20–30 μl risks damaging the pores in the array. ::: (iv) Prepare the flow cell priming mix: add 30 μl of thawed and mixed FLT directly to the tube of thawed and mixed FLB, and mix by pipetting up and down. (v) Load 800 μl of the priming mix into the flow cell via the priming port, avoiding introducing air bubbles. (vi) Wait for 5 min. (vii) Proceed with library dilution for sequencing: thoroughly mix the contents of the LBII tube by pipetting or gently vortexing. ::: warning :warning: The LBII tube contains a suspension of beads. These beads settle very quickly! It is vital that they are mixed **immediately** before use. ::: (viii) Thoroughly mix the contents of SBII (ONT Ligation Kit) and LBII (ONT library loading bead kit) tubes by pipetting. (ix) Prepare the following reaction mix: | Reagent | Volume (μl) per sample | |:-----------:| ------------------------- | | SBII | 37.5 | | LBII | 25.5 | | DNA Library | 12 | (x) Complete the flow cell priming by gently lifting the SpotON sample port cover to make the SpotON sample port accessible. (xi) Load an additional 200 μl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles again by reverse pipetting. :::warning :warning: Do not load the priming mix into the SpotON sample port! ::: (xii) Mix the prepared library gently by pipetting up and down just prior to loading. (xiii) Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. (xiv) Ensure each drop flows into the port before adding the next. The library is loaded dropwise without putting the pipette tip firmly into the port. Avoid introducing any air during pipetting. (xv) Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, then close the priming port and the MinION lid. **B. Preparing and loading the Flongle** (i) Peel back the seal tab until the sample port is exposed. (ii) Prepare the flow cell priming mix in a new PCR tube: add 117 μl of mixed FLB and 3 μl of mixed FLT, mix by pipetting up and down. (iii) Load 120 μl of the priming mix into the sample port, avoiding the introduction of air bubbles by reverse pipetting. (iv) Wait for 5 min. (v) Library dilution for sequencing: thoroughly mix the contents of the LBII tube by pipetting. Critical: The LBII tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use! (vi) Thoroughly mix the contents of SBII (ONT Ligation Kit box) and LBII (ONT library loading bead kit box) tubes by pipetting. (vii) Prepare the following reaction mix: | Reagent | Volume (μl) per sample | |:-----------:| ------------------------- | | SBII | 15 | | LBII | 10 | | DNA Library | 5 | (viii) Mix the prepared library gently by pipetting up and down just prior to loading. (ix) Add 30 μl of sample to the flow cell via the sample port by dialing down the pipetting volume. (x) Gently reseal the sample port with the seal tab, making sure the sample port is sealed properly. (xi) Bring the top (wheel icon section) to its original position, and close the MinION lid. **Getting ready for sequencing** 1. To set up the MinKNOW software, first plug in your MinION device. Critical: From here on, the steps are the same for the standard MinION flow cells and the Flongle flow cell using MinKNOW on a computer. 2. Open MinKNOW on the computer. 3. Select the sequencing device connected to the computer - if using the Mk1C, skip this -, then select the ‘Start Sequencing’ option on the Start homepage. 4. Enter information such as experiment name, sample ID and flow cell type, and select kit type. 5. (Optional) Turn basecalling ‘off’ if you want to basecall the data later on your laptop or a miniserver, or if you plan to sequence for longer than a day. 6. Select the following run options: 24 h or 72 h depending on the maximum run time required, output file fasta5, and save to the local drive (if using Mk1B) or device drive (if using Mk1C).