UWC_1KSA_sea_sponge_metagenome_sequencing_protocol_v1

Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 1 of 10 UWC IMBM Sea Sponge DNA extraction protocol 17/11/2025 Table of Contents SUMMARY ...............................................................................................................................................2 1. REAGENTS REQUIRED.......................................................................................................................... 2 2. EQUIPMENT.......................................................................................................................................... 2 3. CONSUMABLES......................................................................................................................................3 4. PERSONAL PROTECTIVE EQUIPMENT...................................................................................................... 4 5. SAMPLING……………................................................................................................................................4 6. PROTOCOL………………………………………………………………………..………...................................................... 5 7. NOTES………...........................................................................................................................................5 Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 2 of 10 Summary This document describes the method for isolation of intact high-quality DNA from the sea sponge Hymeniacidon perlevis developed during the 1KSA project. The steps for generating sequence data from extracted DNA at the UWC-SCGP DIPLOMICS partner lab, as well as the analysis pipeline, data storage and management plan are shown in Figure 1. Figure 1: 1KSA metagenome sequencing workflow 1. REAGENTS REQUIRED • MetaPolyzyme, DNA free (Merck cat. No. MAC4LDF) • Proteinase K (Merck cat. no. 70663) • T7 endonuclease I (NEB cat. no. M0302S / M0302L) • Lysozyme from chicken egg white (Merck cat. no. 62971) • ZymoBIOMICS DNA Miniprep Kit (cat. no. D4301) • Qiagen REPLI-g UltraFast Mini Kit (25) (cat. no. 150033) DNA E TRACTION AND QC LIBRARY PREPARATION SEQUENCING DATA MANAGEMENT METAGENOME ASSEMBLY LIBRARY PREPARATION RAPID BARCODING KIT DATA MANAGEMENT NAS GLOBUS CONNECT C PC DIRISA DNA E TRACTION AND QC CULTURE BACTERIA E TRACT DNA WIT MAC EREY NAGEL KIT AGAROSE GEL QUBIT SEQUENCING LOAD LIBRARIES WAS AND RELOAD or RUN TO END OF LIFE BASECALLING METAGENOME ASSEMBLY QUALITY CONTROL ASSEMBLY EVALUATION COLLECT SPONGE SAMPLE FILTER SAMPLE E TRACT DNA USING YMO KIT DNA E TRACTION AND QC LIBRARY PREPARATION SEQUENCING DATA MANAGEMENT METAGENOME ASSEMBLY LIBRARY PREPARATION RAPID BARCODING KIT DATA MANAGEMENT NAS GLOBUS CONNECT C PC DIRISA DNA E TRACTION AND QC CULTURE BACTERIA E TRACT DNA WIT MAC EREY NAGEL KIT AGAROSE GEL QUBIT SEQUENCING LOAD LIBRARIES WAS AND RELOAD or RUN TO END OF LIFE BASECALLING METAGENOME ASSEMBLY QUALITY CONTROL ASSEMBLY EVALUATION RUN TO END OF LIFE DNA E TRACTION AND QC LIBRARY PREPARATION SEQUENCING DATA MANAGEMENT METAGENOME ASSEMBLY LIBRARY PREPARATION RAPID BARCODING KIT DATA MANAGEMENT NAS GLOBUS CONNECT C PC DIRISA DNA E TRACTION AND QC CULTURE BACTERIA E TRACT DNA WIT MAC EREY NAGEL KIT AGAROSE GEL QUBIT SEQUENCING LOAD LIBRARIES WAS AND RELOAD or RUN TO END OF LIFE BASECALLING METAGENOME ASSEMBLY QUALITY CONTROL ASSEMBLY EVALUATION Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 3 of 10 • Qubit dsDNA Quantitation, HS reagent kit (buffer and fluorescent dye ThermoFisher cat. no. Q32854) • Molecular grade ethanol (Sigma cat. no. 1.08543) • DNase/RNase-Free Deionized Water (ThermoFisher cat. no. A57775) • Agarose, SeaKem LE (Lonza cat. no. 50004) • 3m Sodium (Na) acetate pH5.2 2. EQUIPMENT • Pipettes (P1000, P200, P20, P10) • Centrifuge to spin 50 mL tubes @ 5000 X g • Centrifuge to spin 2 mL tubes @ 5000 / 11000 X g • Water bath • Qubit fluorometer • Pipette bulb / aid / pump • Small air compressor pump (AirCraft Pneumatic products cat. no. SG COMP04) • Tubing to connect pump to Nalgene Reusable Bottle Top Filter • Nalgene Reusable Bottle Top Filter (ThermoFisher cat. no. DS0320-5045) • Light microscope • Thermocycler 3. CONSUMABLES • 1.5 mL / 2mL DNA LoBind Eppendorf tubes • 200µL DNA LoBind Eppendorf PCR tubes • 50 mL conical tubes • P1000 tips • P200/P20 tips • P10 tips • 20ml syringes • 10ml sterile serological pipettes Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 4 of 10 • Sartorius 47mm filter 1.2 µm (cat. no. 12303--47------N) • Sartorius 47mm filter 0.8 µm (cat. no. 11104--47------N) • Merck Millipore 2.0 µm glass fiber syringe filter (cat. no. Millex-AP SLAP05010) • Merck Millipore MCE Membrane Filter, 0.025 μm Pore Size (cat. no. VSWP02500) • Microscope slides and cover slips 4. PERSONAL PROTECTIVE EQUIPMENT • Observe standard best practice for laboratory safety • Closed shoes • Safety goggles • Nitrile gloves • Lab coat 5. SAMPLING • Ideally the sample material should be as fresh as possible so set time aside to ensure that the sample is collected and processed as soon as possible • Prepare a clean autoclaved 500ml Schott bottle. For smaller sampling volumes, use 50ml conical tubes • Wrap a funnel in aluminum foil and autoclave. Wrap metal spatulas in foil and autoclave. • Take a spray bottle of 70% ethanol, nitrile gloves and a roll of paper towel • Take a notepad, pen and marker pens if needed The sponge used in this case contains a large amount of spongocoel / mesohyl fluid, so extracting this liquid with the associated bacteria is trivial but may be more difficult for other sponge species. After putting on gloves, the funnel is placed into the mouth of the sterilized Schott bottle to collect the liquid squeezed from the sponge. The sponge material is scraped from the rock face using a sterile spatula and squeezed / macerated above the funnel to collect the liquid. One has to be quite forceful to extract the maximum fluid from the sponge and using two spatulas to squeeze the material between might be helpful. The sponge contains comparatively high amounts of microbial biomass and not much material is needed to have enough for DNA extraction and sequencing. We would recommend not collecting more than 200ml spongocoel / mesohyl fluid per sampling trip. Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 5 of 10 6. PROTOCOL • Sample pre-processing 1) Transfer collected material to 50ml conical tubes and perform a low-speed centrifugation (1000 RPM for 5min) to remove very large debris 2) We use a sterile 10ml pipette and pipette bulb / aid / pump to remove the supernatant from the tube and transfer to a new tube. Do not decant the supernatant as the pellet is still quite loose and a portion of this will end up sliding down with the supernatant. Sacrifice several milliliters to avoid taking any of the pellet with. 3) The supernatant is filtered, first through a 2.0 µm glass fiber syringe filter (Millex-AP SLAP05010) using a 20ml syringe (Figure 2A) 4) Filter through 1.2µm (cat. no. 12303--47------N) 47mm filter using (ThermoFisher cat. no. DS0320-5045) filtration unit and AirCraft air compressor pump. 5) Filter through 0.8µm (11104--47------N) 47mm filter using (ThermoFisher cat. no. DS0320-5045) filtration unit and AirCraft air compressor pump. Figure 2: A) Sample prior to (left hand tube) and following (right hand tube) syringe driven filtration through a 2.0µm glass fiber filter. B) Sample prior to (left hand tube) and following (right hand tube) pump driven filtration through a 1.2µm followed by a 0.8µm cellulose acetate filters. Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 6 of 10 6) Centrifuge sample at 5000 RPM for 5min to yield a pellet called the “low speed” fraction. Remove supernatant to tubes suitable to centrifuge at 13000 RPM. 7) Centrifuge supernatant at 13000 RPM for 5min to yield a pellet called the “high speed” fraction. 8) Discard the supernatant of both fractions and wash both pellets twice in 1ml filter sterilized sea water (resuspend each and centrifuge at the appropriate speed to recover the cells). 9) Following the second wash step the low speed and high-speed pellets are combined in 400µl sterile sea water. 10) If the sample is not to be pre-processed with an enzyme, add 1.2 ml DNA/RNA shield and proceed to process it through the ZymoBIOMICS DNA Miniprep Kit (D4301). 11) If the sample is to be pre-processed with just Proteinase K, add 1.2 ml DNA/RNA shield, add Proteinase K to a final concentration of 1mg/ml and incubate at 55°C for 3 hours to overnight. 12) If treating the cells with either Lysozyme (1mg/ml final concentration) or Metapolyzyme (50µl of the stock in 400µl) prior to Proteinase K treatment, add the enzyme first, and incubate according to manufacturer’s recommendations. Following enzyme pre-treatment, add 1.2ml DNA/RNA shield then add Proteinase K and incubate at 55°C for 3 hours to overnight. 13) After any pre-treatment, process the ~1.6 ml sample through the ZymoBIOMICS DNA Miniprep Kit (D4301). Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 7 of 10 Using the appropriately filtered material described above, we performed four test DNA isolations: 1) No pretreatment, 2) Proteinase K only pretreatment, 3) Lysozyme pretreatment followed by Proteinase K and 4) Metapolyzyme pretreatment followed by Proteinase K (Figure 3). Figure 3: Final DNA isolation attempt using pressure driven filtration. This demonstrated that material treated in this fashion resulted in successful extraction of relatively high amounts (30ng/µl for a total of ~900ng in a 30µl elution) per amount of biomass. In the past this has been a signature of successful bacterial genome and metagenomic extraction in particular from this sponge material. If enough DNA is recovered directly from the sponge material, one could proceed to create a sequencing library using your preferred kit (SQK-RAD114 / SQK-RBK114.24/96 / SQK-LSK114). In this regard, we’ve exclusively used rapid sequencing kits or rapid barcoding kits with the understanding that this limits output and reduces read length N50 values. If too little DNA has been recovered, or you want improved basecalling, you can continue from step 14 below. 14) Next, perform whole genome amplification (WGA) using the Qiagen REPLI-g UltraFast Mini Kit (25) (cat. no. 150033) according to the manufacturer’s recommendations on DNA preparations. This ensures Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 8 of 10 sufficient DNA for library creation as well as improved data generation through replacement of DNA modifications with canonical nucleotides. 15) Following WGA, precipitate DNA through the addition of 1/10 volume 3M Na-acetate pH5.2, and two volumes 100% molecular grade ethanol. Invert the tube several times to ensure proper mixing and observe DNA precipitate forming. Centrifuge at 13000 RPM for 10min at 4°C. Invert the tube on paper towel to drain the ethanol and air dry the pellet. Resuspend in 20µl molecular grade water. Alternatively, the DNA can be cleaned by using AMPure XP beads according to the Ligation sequencing gDNA V14 - whole genome amplification (SQK-LSK114) (WAL_9192_v114_revK_25Sep2025) protocol. 16) If DNA was precipitated it may be useful to dialyze the DNA to remove excess salt. This is done by floating a VSWP02500 filter on molecular grade water in a petri dish and carefully pipetting the resuspended DNA onto the filter. The DNA is left to dialyze for up to 2 hours after which is removed by pipetting. 17) At this point a decision needs to be made depending on which library prep kit is to be used: 1) We have successfully used rapid sequencing kits (SQK-RAD114) or rapid barcoding kits (SQKRBK114.24/96), to sequence metagenomic DNA prepared as described above without the pores suffering massively reduced performance, however this requires that the tagmentation step work well as this fragments the DNA (acts like T7 Endonuclease I). 2) Alternatively, the DNA can be digested with T7 Endonuclease I and used as is together with a rapid sequencing kit or rapid barcoding kit to create a sequencing library, however the read length N50 might be reduced. 3) The DNA can be digested with T7 Endonuclease I and the DNA ends repaired according to Ligation sequencing gDNA V14 - whole genome amplification (SQK-LSK114) (WAL_9192_v114_revK_25Sep2025) protocol, to then create a library using the SQK-LSK114 kit. This would ensure highest output from the chosen flow cell and the highest read N50 values. Institute for Microbial Biotechnology and Metagenomics Core2, Life Science Building, university of the Western Cape, Robert Sobukwe rd, Bellville, Cape Town, South Africa, 7535 Tel: +27 21 959 2325 Email: [email protected], [email protected]; www.imbm.co.za Page 9 of 10 18) Digest the DNA using T7 Endonuclease I according to the Oxford Nanopore Technologies Ligation sequencing gDNA V14 - whole genome amplification (SQK-LSK114) (WAL_9192_v114_revK_25Sep2025) protocol. Combine the reagents in the table below and incubate the reaction for 60 minutes at 37°C. 19) Start at step 29 of the “Whole genome amplification” section of the Ligation sequencing gDNA V14 - whole genome amplification (SQK-LSK114) (WAL_9192_v114_revK_25Sep2025) protocol to complete sequence library preparation. • The annotated draft genome assembly files are transferred to the “DIRISA Storage CPT” servers for long term storage. Reagent Volume 1.5µg of amplified DNA (from previous step) 24 µl NEBuffer 2 3 µl T7 Endonuclease I 3 µl Total 30 µl