Good Morning All,
I am trying to sequence a few hard-to-ID Enterobacteria using the Oxford Nanopore Rapid Barcoding Kit (SQK-RBK004) on the MinION. These samples have already been sequenced on an Illumina Miseq.Through trial and error I've found that ONT's promise of 12x multiplexing is...optimistic at best. To maximize reads I've cut multiplexing down to 2x and seem to get decent reads with good quality (for ONT), although even still I can't seem to get good read-equity between samples. One might have 1.2Gbp called while the other has 1/4th that number.
My workflow is slightly modified from the base protocol as follows:
-Pre-Library-Prep Bead concentratio using Axygen Ampure beads. (1:1 Ratio with 30 minute incubation + 30 min 37C Elution). Our samples are not nearly as concentrated as needed for the protocol so this is a must for library prep to even have a hope of working.
-2x initial rxn volume (15uL gDNA input, 5uL Fragmentation Barcode)
(10uL was too small to ensure volume sufficient for Qubit / Tape Station QC)
-Post-Library-Prep Bead Concentration to pool samples to the 10uL volume required for loading the final library.
For pooling, I've followed the approach that we use for our Illumina runs.
-Qubit fluorometer to get concentration
-Agilent tape station to give fragment size
-Dilution excel sheet to calculate how much volume is needed to ensure an equimolar amount between samples.
So far it seems as though my attempts at normalizing my PAL are a crap-shoot with no real correlation between my efforts and read equity. This is a PCR-Free protocol, so perhaps the lack of a single amplified fragment size is throwing my assumptions of "Average Fragment length" out the window.
Anyone have any suggestions? Protocols? Suggestions? Experienced something similar?
Thanks!
I am trying to sequence a few hard-to-ID Enterobacteria using the Oxford Nanopore Rapid Barcoding Kit (SQK-RBK004) on the MinION. These samples have already been sequenced on an Illumina Miseq.Through trial and error I've found that ONT's promise of 12x multiplexing is...optimistic at best. To maximize reads I've cut multiplexing down to 2x and seem to get decent reads with good quality (for ONT), although even still I can't seem to get good read-equity between samples. One might have 1.2Gbp called while the other has 1/4th that number.
My workflow is slightly modified from the base protocol as follows:
-Pre-Library-Prep Bead concentratio using Axygen Ampure beads. (1:1 Ratio with 30 minute incubation + 30 min 37C Elution). Our samples are not nearly as concentrated as needed for the protocol so this is a must for library prep to even have a hope of working.
-2x initial rxn volume (15uL gDNA input, 5uL Fragmentation Barcode)
(10uL was too small to ensure volume sufficient for Qubit / Tape Station QC)
-Post-Library-Prep Bead Concentration to pool samples to the 10uL volume required for loading the final library.
For pooling, I've followed the approach that we use for our Illumina runs.
-Qubit fluorometer to get concentration
-Agilent tape station to give fragment size
-Dilution excel sheet to calculate how much volume is needed to ensure an equimolar amount between samples.
So far it seems as though my attempts at normalizing my PAL are a crap-shoot with no real correlation between my efforts and read equity. This is a PCR-Free protocol, so perhaps the lack of a single amplified fragment size is throwing my assumptions of "Average Fragment length" out the window.
Anyone have any suggestions? Protocols? Suggestions? Experienced something similar?
Thanks!
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