Hi all,
I've been having this issue that has been plaguing my PhD. Some of my sequencing runs come back with terrible uneven read coverage. I am guessing it is a pooling problem but still not 100% sure.
For some background on my pipeline: after my final bead clean step I quantify all my samples and usually get a huge range of concentrations from 5ng/ul to 100+ ng/ul. This isn't surprising as my gut microbiome samples come from a variety of fish species. I then proceed to pool the samples by adding different volumes to create a 20ng/ul final pooled library. Theoretically, I am adding the same amount of moles of each sample to the pool. Obviously it isn't perfect but I do not understand why my read coverage is so terrible. I usually get back many samples with <1000 reads and some with over 300k reads on an Ilumina Miseq (16S V4 amplicon).
My question is: is there a different way to normalize the concentrations before pooling?
Is diluting samples with water to normalize concentrations (adding different amount of H20 to get them all the same concentration) helpful before pooling?
Are highly variable sample concentrations prior to pooling a culprit for uneven read coverage?
For indexing (2nd PCR), I usually add 1ul of PCR product or 2ul depending on the brightness from the gel. This has worked fine before but would it help if I quantified my PCR products and then added a more precise volume for indexing to keep it more normalized?
I pool in a way so I am adding a minimum of 2ul so as to avoid pipette error with small volumes.
Some additional info:
I do a 2-step PCR process with 515-806R primers
Qiagen multiplex kit for PCR 1 and Kappa Hi-Fi for PCR 2
Nextera Unique Dual indexes for indexing
Omega RXN Pure Beads for bead cleans
Qubit for quantification
Any help would much be appreciated!!
Cheers,
Sam
I've been having this issue that has been plaguing my PhD. Some of my sequencing runs come back with terrible uneven read coverage. I am guessing it is a pooling problem but still not 100% sure.
For some background on my pipeline: after my final bead clean step I quantify all my samples and usually get a huge range of concentrations from 5ng/ul to 100+ ng/ul. This isn't surprising as my gut microbiome samples come from a variety of fish species. I then proceed to pool the samples by adding different volumes to create a 20ng/ul final pooled library. Theoretically, I am adding the same amount of moles of each sample to the pool. Obviously it isn't perfect but I do not understand why my read coverage is so terrible. I usually get back many samples with <1000 reads and some with over 300k reads on an Ilumina Miseq (16S V4 amplicon).
My question is: is there a different way to normalize the concentrations before pooling?
Is diluting samples with water to normalize concentrations (adding different amount of H20 to get them all the same concentration) helpful before pooling?
Are highly variable sample concentrations prior to pooling a culprit for uneven read coverage?
For indexing (2nd PCR), I usually add 1ul of PCR product or 2ul depending on the brightness from the gel. This has worked fine before but would it help if I quantified my PCR products and then added a more precise volume for indexing to keep it more normalized?
I pool in a way so I am adding a minimum of 2ul so as to avoid pipette error with small volumes.
Some additional info:
I do a 2-step PCR process with 515-806R primers
Qiagen multiplex kit for PCR 1 and Kappa Hi-Fi for PCR 2
Nextera Unique Dual indexes for indexing
Omega RXN Pure Beads for bead cleans
Qubit for quantification
Any help would much be appreciated!!
Cheers,
Sam
Comment