TITLE: DNA extraction and genomic sequencing library preparation for individual root-knot nematodes
AUTHORS: Graham S Sellers, Dave Lunt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A simple, high molecular weight DNA extraction and long read genomic sequencing library preparation from individual root-knot nematodes (RKN). This protocol successfully sequences juvenile stage 2 (j2) RKN individuals, which are less than 500 µm long, and therefore the protocol is applicable for other meiofauna.</div><div class = "text-block">DNA extraction follows a highly modified Mu-DNA: Tissue protocol (Sellers et al., 2018) using a solid phase reversible immobilization (SPRI) magnetic bead capture method, adapted from Rohland and Reich, (2012). Library preparation follows Oxford Nanopore Technologies' Rapid PCR Barcoding Kit (SQK-RPB004), modified for extremely low input DNA template and converted for Flongle flow cell sequencing.</div><div class = "text-block">The method is capable of generating sufficient reads for successful taxonomic assignment from juvenile stage 2 (j2) to adult individuals using our bioinformatics workflow available at:</div><div class = "text-block"><a href="https://github.com/Graham-Sellers/RKN_genomic_taxonomic_assignment" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/Graham-Sellers/RKN_genomic_taxonomic_assignment</span></a><span style = ":;">.</span></div><div class = "text-block">Rohland N, Reich D (2012) Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome research 22:939–946.</div><div class = "text-block"><span>Sellers GS, Di Muri C, Gómez A, Hänfling B (2018) Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types. Metabarcoding and Metagenomics 2:e24556. (</span><a href="https://dx.doi.org/10.17504/protocols.io.qn9dvh6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.qn9dvh6</span></a><span>)</span></div></div>

[STEPS]
?. [DNA extraction]
SPRI based DNA extractionLysisAdd 200 μl Lysis Master Mix to 1.5 ml LoBind tube containing nematode sample. Vortex briefly to mix and centrifuge tube for 1 secPlace in Thermomixer at 55°C for 90 mins at 550 rpm.Centrifuge tube for 1 secInhibitor removalAdd 70 μl (0.3 X volume) of Flocculant Solution, invert several times to mix and incubate on ice for a minimum of 10 minCentrifuge at ≥ 10,000 xg for 2 min at room temperatureWithout disturbing the pellet, transfer supernatant to a fresh 1.5 ml LoBind tubeSPRI DNA bindingAdd 400 μl (2 X volume) of DNA Extraction Bead Solution. Place on HulaMixer (continual rotation) for 10 minPlace on magnetic stand until supernatant is clear and beads are bound towards magnetWhile on the stand carefully remove and discard supernatant without disturbing beadsWashAdd 1000 μl Wash Solution. Incubate at room temperature for 30 secsWhile on the stand carefully remove and discard supernatant without disturbing beadsRepeat steps 11 to 12 a further timeCentrifuge tube for 1 sec. Place back on magnetic stand ensuring beads are bound towards magnet. Remove all remaining Wash Solution with a 10 μl pipette. Air dry tube with cap open until beads are completely dry (i.e. no longer shiny)ElutionAdd 25 μl Elution Buffer (55°C) and gently flick to resuspend beads. Ensure all beads are resuspended with no clumps. Centrifuge tube for 1 secPlace in Thermomixer at 55°C for 10 mins at 550 rpm. Centrifuge tube for 1 secPlace on magnetic stand until supernatant is clear and beads are bound towards magnetCarefully transfer eluate to a fresh 1.5 ml LoBind tube without disturbing beadsInhibitor removalAdd 70 μl (0.3 X volume) of Flocculant Solution, invert several times to mix and incubate on ice for a minimum of 10 minCentrifuge at ≥ 10,000 xg for 2 min at room temperatureWithout disturbing the pellet, transfer supernatant to a fresh 1.5 ml LoBind tubeSPRI DNA bindingAdd 400 μl (2 X volume) of DNA Extraction Bead Solution. Place on HulaMixer (continual rotation) for 10 minPlace on magnetic stand until supernatant is clear and beads are bound towards magnetWhile on the stand carefully remove and discard supernatant without disturbing beadsWashAdd 1000 μl Wash Solution. Incubate at room temperature for 30 secsWhile on the stand carefully remove and discard supernatant without disturbing beadsRepeat steps 11 to 12 a further timeCentrifuge tube for 1 sec. Place back on magnetic stand ensuring beads are bound towards magnet. Remove all remaining Wash Solution with a 10 μl pipette. Air dry tube with cap open until beads are completely dry (i.e. no longer shiny)ElutionAdd 25 μl Elution Buffer (55°C) and gently flick to resuspend beads. Ensure all beads are resuspended with no clumps. Centrifuge tube for 1 secPlace in Thermomixer at 55°C for 10 mins at 550 rpm. Centrifuge tube for 1 secPlace on magnetic stand until supernatant is clear and beads are bound towards magnetCarefully transfer eluate to a fresh 1.5 ml LoBind tube without disturbing beadsSPRI DNA bindingAdd 400 μl (2 X volume) of DNA Extraction Bead Solution. Place on HulaMixer (continual rotation) for 10 minPlace on magnetic stand until supernatant is clear and beads are bound towards magnetWhile on the stand carefully remove and discard supernatant without disturbing beadsWashAdd 1000 μl Wash Solution. Incubate at room temperature for 30 secsWhile on the stand carefully remove and discard supernatant without disturbing beadsRepeat steps 11 to 12 a further timeCentrifuge tube for 1 sec. Place back on magnetic stand ensuring beads are bound towards magnet. Remove all remaining Wash Solution with a 10 μl pipette. Air dry tube with cap open until beads are completely dry (i.e. no longer shiny)ElutionAdd 25 μl Elution Buffer (55°C) and gently flick to resuspend beads. Ensure all beads are resuspended with no clumps. Centrifuge tube for 1 secPlace in Thermomixer at 55°C for 10 mins at 550 rpm. Centrifuge tube for 1 secPlace on magnetic stand until supernatant is clear and beads are bound towards magnetCarefully transfer eluate to a fresh 1.5 ml LoBind tube without disturbing beadsWashAdd 1000 μl Wash Solution. Incubate at room temperature for 30 secsWhile on the stand carefully remove and discard supernatant without disturbing beadsRepeat steps 11 to 12 a further timeCentrifuge tube for 1 sec. Place back on magnetic stand ensuring beads are bound towards magnet. Remove all remaining Wash Solution with a 10 μl pipette. Air dry tube with cap open until beads are completely dry (i.e. no longer shiny)ElutionAdd 25 μl Elution Buffer (55°C) and gently flick to resuspend beads. Ensure all beads are resuspended with no clumps. Centrifuge tube for 1 secPlace in Thermomixer at 55°C for 10 mins at 550 rpm. Centrifuge tube for 1 secPlace on magnetic stand until supernatant is clear and beads are bound towards magnetCarefully transfer eluate to a fresh 1.5 ml LoBind tube without disturbing beadsElutionAdd 25 μl Elution Buffer (55°C) and gently flick to resuspend beads. Ensure all beads are resuspended with no clumps. Centrifuge tube for 1 secPlace in Thermomixer at 55°C for 10 mins at 550 rpm. Centrifuge tube for 1 secPlace on magnetic stand until supernatant is clear and beads are bound towards magnetCarefully transfer eluate to a fresh 1.5 ml LoBind tube without disturbing beads
Before beginning DNA extractionThaw samples on ice. Centrifuge 1.5 ml Eppendorf LoBind tube containing nematode sample at ≥ 10,000 xg for 1 min to ensure sample is at bottom of tube. Pipette off excess water if sample is stored in water, be careful not to remove sample. A small volume of water remaining (~10 μl) will not affect extraction.Take DNA Extraction Bead Solution from the fridge and allow it to reach room temperature. Vortex to mix thoroughly prior to use.Create Lysis Master Mix: 730 μl Lysis Solution, 250 μl Tissue Lysis Additive and 60 μl PK. Vortex to mix. Incubate at 55°C until required.Incubate Elution Buffer at 55°C until required.
Store DNA at -20°C until required.
?. [Library preparation and sequencing]
Library preparation and Sequencing
Library preparation and sequencing is broken down into distinct stages. At the end of the first two stages are suitable stopping points. This allows the process to be carried out over multitiple days if required.
?. [Library preparation and sequencing]
DNA fragmentation/tagmenting and PCR barcodingDNA fragmentation/tagmentingAdd appropriate sample volume to 1 μl FRM in a 0.2 ml PCR tube, close lid and flick gently to mix. Centrifuge briefly to collect droplets from lid. Place on ice until required.In a thermal cycler, incubate PCR tube for: 1 min @ 30°C, then 1 min @ 80°C, hold at 4°C. DNA template is now tagmented, place on ice until required.PCR BarcodingMake a PCR master mix per sample (allow ~5% for pipetting error):20 μl Nuclease-free water25 μl LongAmp Taq 2X master mixAdd 45 μl PCR master mix to each tagmented sample and 1 μl of RLB (01-12a). Flick gently to mix, centrifuge briefly to collect droplets from lid. Place on ice until required.
Before beginning fragmentation/tagmenting and PCR barcodingThaw samples on ice. Thaw Rapid PCR Barcoding Kit reagents: Barcodes (RLB 01-12a) at room temperature and place Fragmentation Mix (FRM) on ice. Keep reagents on ice until required, vortex briefly and spin down prior to use.DNA quantificationQuantify 2 μl of each sample on a Qubit 3.0 fluorometer using high-sensitivity (HS) dsDNA assay.
Important: If sample DNA concentration is less than 1 ng/μl (or ‘too low’) use 5 μl of sample for fragmentation, otherwise use 3 μl. Maximum amount of DNA to be used is 5 ng (as per Oxford Nanopore Rapid PCR Barcoding Kit: SQK-RPB004).
Thermal cycler conditionsIf initial sample DNA template > 0.1 ng/μl, place sample in a thermal cycler with:3 mins @ 95°C, 15 X (15 secs @ 95°C, 15 secs @ 56°C, 6 mins @ 65°C), 6 mins @ 65°C, hold at 4 °CElse, place sample in a thermal cycler with:3 mins @ 95°C, 20 X (15 secs @ 95°C, 15 secs @ 56°C, 6 mins @ 65°C), 6 mins @ 65°C, hold at 4 °C
Stopping point: PCR barcoded samples can be stored in a fridge overnight or up to a week if required.
?. [Library preparation and sequencing]
Sample concentrationAdd 50 μl (1 X volume) of Library Prep Bead Solution to each sample. Mix by pipettingLeave to stand at room temperature for 10 minsPlace on magnetic stand until supernatant is clear and beads are bound towards magnetWhile on the stand carefully remove and discard supernatant without disturbing beadsAdd 150 μl Wash Solution. Incubate at room temperature for 30 secsWhile on the stand carefully remove and discard supernatant without disturbing beadsRepeat steps 11 to 12 a further timeCentrifuge tube for 1 sec. Place back on magnetic stand ensuring beads are bound towards magnet. Remove all remaining Wash Solution with a 10 μl pipette. Air dry tube with cap open until beads are completely dry (i.e. no longer shiny)Add 15 μl Library Buffer (55°C) and gently flick to resuspend beads. Ensure all beads are resuspended with no clumps. Centrifuge tube for 1 secLeave to stand at room temperature for 10 minsPlace on magnetic stand until supernatant is clear and beads are bound towards magnetCarefully transfer eluate to a fresh 0.2 ml PCR tube without disturbing beads
Before beginning sample concentrationA magnetic stand or rack is required for this step. If it cannot accommodate 0.2 ml PCR tubes, transfer each PCR barcoded sample to a 1.5 ml Eppendorf DNA LoBind tube.Take Library Prep Bead Solution from the fridge and allow it to reach room temperature. Vortex to mix thoroughly prior to use.Create Lysis Master Mix: 730 μl Lysis Solution, 250 μl Tissue Lysis Additive and 60 μl PK. Vortex to mix. Incubate at 55°C until required.Incubate Library Buffer at 55°C until required.
Stopping point: Concentrated samples can be stored in a fridge overnight or up to a week if required.
?. [Library preparation and sequencing]
Sample DNA quantification and molarity estimationDNA quantification Quantify 2 μl of each concentrated sample on a Qubit 3.0 fluorometer using high-sensitivity (HS) dsDNA assay.Fragment length estimationFor sample DNA fragment length estimates there are three options we have used:Molarity estimationBased on the sample quantification and fragment length estimates (whichever option chosen), the molarity of each sample can now be calculated in fmol/μl. For this use the following equation:Pool samplesSamples should be pooled as equimolar as possible for best results. However, this is not entirely necessary or sometimes even possible. Aim for a final library concentration of ~ 2 fmol/μl. This will mean that the 5 μl volume of library used for sequencing a full 12 samples will contain 5 - 10 fmol of DNA, ideal for Flongle sequencing.
Option 1 (ideal and accurate but expensive, does not work with low DNA concentration):1 μl of each sample was measured on a Tapestation 2200 (Agilent Technologies) using genomic tape.Option 2 (cheap but less accurate, may not work with low DNA concentration):2 μl of each sample was run on a 1% agarose gel against 0.5 μl Generuler 1 Kb DNA ladder (or similar, diluted 1:20). Estimate fragment length based on gel imaging.Option 3 (free but less accurate, DNA concentration not an issue):Assume all samples have a fragment length of 7 Kb.
fmol/μl = DNA concentration / ((DNA fragment length X 1,000 X 617.96) + 36.04) X 1,000,000DNA concentration is in ng/μl, DNA fragment length is in Kb.
It is perfectly feasible to concentrate (see sample concentration above) or dilute (using Library Buffer) the pooled library as required. After adjusting the concentration it is advised to quantify 2 μl of the pooled library on a Qubit 3.0 and recalculate molarity (as above) using the average fragment length.
?. [Library preparation and sequencing]
Sequencing adapter ligationAllow Rapid Adapter (RAP) to reach room temperature. Vortex briefly and spin down.Transfer 5 µl of final library into a 1.5 ml Eppendorf DNA LoBind tubeAdd 0.5 µl RAP to the tube. Flick gently to mix and spin down brieflyIncubate for 5 - 10 minutes at room temperaturePlace on ice until requiredLoading the Flongle flow cellFollow any Flongle protocol from ‘Loading the Flongle flow cell’ (e.g. this protocol).SequencingWe recommend specifying a directory for sequencing output (i.e. a desktop or external hard drive directory) rather than the hard-to-find default output directory of ONT’s MinKNOW software. Disable all base calling options - we basecall the output fast5s with Guppy GPU HAC post sequencing. Run the sequencing for the default 24 hours. A longer sequencing period may allow for more data, however, we have found reads begin to plateau around the 24 hour mark using our protocol.
Note: It is important to perform a flow cell check prior to loading to ascertain the flow cell’s quality.