Library Prep and Sequencing

Follow universal lab safety precautions: wear a lab coat and gloves when working in the lab, no open-toed shoes, no eating and drinking.

Ocean and Sewage Samples

Library Preparation of ocean and sewage samples is done with the SQK-LSK114 kit from Oxford Nanopore. One sample per flow cell.

The protocol is from ONT; libraries are prepared and sequenced as written with 1 ug DNA:

Sequencing is done on flow cell version R10.4.1 flow cells (FLO-MIN114) using a MinION Mk1B.

Extra Reagents for SQK-LSK114

Per Library

Reagent	Catalog	Kit Volume	Volume 1X Library	Library/Kit	For 6 Libraries	Price	Price Per Sample
NEBNext FFPE Repair Mix Enzyme	NEB M6630	48 ul	2 ul	24	1 kit	161	6.71
Ultra II End Prep Enzyme Mix	NEB E7546	72 ul	3 ul	24	1 kit	218	9.08
NEB Next Quick T4 Ligase	NEB E6056	100 ul	10 ul	10	1 kit	314	31.40
BSA at 50 mg/ml	Invitrogen AM2616	1 ml	5 ul	200	1 kit	167	0.84
Ampure XP	Homemade	NA	60 + 40 ul	NA	600 ul		0.10
Ethanol	NA	NA	500 ul	NA	500 ul		0.50
Ligation Kit	ONT SQK-LSK114	NA	NA	6	1	690	115.00
Flow Cell	ONT	NA	NA	12	12	8100	675
						TOTAL	838.63

Library Preparation of bacterial isolates is done with the SQK-NBD114.24 kit from Oxford Nanopore. Up to 10 samples per flow cell.

The protocol is from ONT; libraries are prepared and sequenced as written with 400 ng DNA:

Sequencing is done on flow cell version R10.4.1 flow cells (FLO-MIN114) using a MinION Mk1B.

Extra Reagents for SQK-NBD114.24

Per Isolate

Reagent	Catalog	Kit Volume	Volume 1X Isolate	Isolates/Kit	For 50 Isolates	Price	Price/Sample
NEBNext FFPE Repair Mix Enzyme	NEB M6630	48 ul	0.5 ul	96	1 kit	161.00	1.68
Ultra II End Prep Enzyme Mix	NEB E7546	72 ul	0.75 ul	96	1 kit	218.00	2.27
Blunt/TA Ligase Master Mix	NEB M0367	250 ul	10 ul	25	3 kits	93	3.72
Ethanol	NA	NA	400 ul	NA	20 ml	NA	0.50
						TOTAL	8.17

Per Library