Hello,
I am using Agilent's Sureselect human all exon v4 kit to make libraries for Illumina sequencing.
Experiment details:
Using version 1.3 protocol
Covaris S2 used for shearing
Incubations are carried out on a tube heat block
Ampure XP beads are used for sample clean ups
MJ Research dyad PCR blocks are used for the amplification steps
RNase free eppendorf tubes with filter tips are used throughout
Agilent 2100 bioanalyser instrument used
In the literature, it says that the expected on target reads is between 70-80%, but after analyses we are only getting around 45-60% for most of our libraries. A great number of our reads are even as low as 28% on target. I have attached a document with all the Agilent traces for one library prep which gave 28% bases on target after final analyses.
Can anyone advise on why this may be occurring?
For example, could there be a minimum amount of library needed after each step? I know that 500ng is needed before hybridisation but what about other steps? The reason I'm asking is that some of the Agilent traces show lower peaks than others and I was wondering if this affects the final quantity.
Thanks in advance for your help,
Anna.
I am using Agilent's Sureselect human all exon v4 kit to make libraries for Illumina sequencing.
Experiment details:
Using version 1.3 protocol
Covaris S2 used for shearing
Incubations are carried out on a tube heat block
Ampure XP beads are used for sample clean ups
MJ Research dyad PCR blocks are used for the amplification steps
RNase free eppendorf tubes with filter tips are used throughout
Agilent 2100 bioanalyser instrument used
In the literature, it says that the expected on target reads is between 70-80%, but after analyses we are only getting around 45-60% for most of our libraries. A great number of our reads are even as low as 28% on target. I have attached a document with all the Agilent traces for one library prep which gave 28% bases on target after final analyses.
Can anyone advise on why this may be occurring?
For example, could there be a minimum amount of library needed after each step? I know that 500ng is needed before hybridisation but what about other steps? The reason I'm asking is that some of the Agilent traces show lower peaks than others and I was wondering if this affects the final quantity.
Thanks in advance for your help,
Anna.
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