eDNA sediment SMS3
The COI gene amplicons were purified using 1.6 NucleoMag (Macherey-Nagel, Germany) beads. The purified amplicons were quantified and used to prepare the library using Ligation sequencing amplicons - Native Barcoding Kit (SQK-LSQ109 with EXP-NBD104) (Oxford Nanopore Technologies, UK). 100-200 fmol of purified amplicons from each sample was end-repaired by NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, USA) and further cleaned using 1 NucleoMag beads. Subsequently, the purified amplicons were ligated with Native Barcodes using Blunt/TA Ligase Master Mix (New England Biolabs, USA) and cleaned again with 1 NucleoMag beads. Subsequently, the barcoded samples were pooled in equimolar concentrations and ligated with sequencing adaptor (AMII) using NEBNext Quick T4 DNA Ligase (New England Biolabs, USA). The library was further cleaned using 0.4 NucleoMag Select beads and finally eluted into 15 l of elution buffer. Final library yield was quantified using Qubit dsDNA HS assay kit (Invitrogen, Thermo Fisher Scientific Inc., USA). The adapter ligated, barcoded library was loaded into a SpotON flow cell R9.4 (FLO-MIN106) on a MinION Mk1B device (Oxford Nanopore Technologies, Oxford, UK) and sequencing was performed using MinKNOW v22.08.9 in an 8 hrs sequencing run.