Hello to all of you.
I am a newbie in the genomics field, so I address here my problem hoping for some feedback and solving ideas.
I am developing a project for 16S V4 region sequencing to understand the diversity of bacterial community in fish intestines. I use feces and mucus as sample, extract genomic DNA with column purification (kit specific for stool) and after quantify prepare the library with NextFlex kit from Bioscientific. I optimized the PCR cycle number and the template concentration to amplify, and I got acceptable amplicon concentrations (measured by Qubit). The problem starts when I check on a gel or by bioanalyzer I will always have (or almost always) 2 bands/peaks) (1 at 300 bp and other at 400bp). The target amplicon should have about 300 bp so I did a gel purification of the target band but then the concentrations are very low and MiSeq doesn't detect signal and fails to clusterize. I asked BioScientific tech help and they reply it should be from any kind of eukaryotic cells around, but I still think it should not derive in two bands but one.
The options I thought were to prepare a first PCR with universal primers (the same of the kit without illumina adapters) and used as template for the kit library prep, or gel band purification, and I was advice to go with the gel band purification. I did and its always too low yield for Miseq to recognize even with PhiX control added.
So I was wondering if anybody has a clue of what might be happening and any idea how to overpass this. Increasing DNA template concentration is no good since it reaches some kind of limit for the PCR of the kit and does not bring anything new in therms of concentration.
I thank you in advance and sorry for the long text.
Ana
I am a newbie in the genomics field, so I address here my problem hoping for some feedback and solving ideas.
I am developing a project for 16S V4 region sequencing to understand the diversity of bacterial community in fish intestines. I use feces and mucus as sample, extract genomic DNA with column purification (kit specific for stool) and after quantify prepare the library with NextFlex kit from Bioscientific. I optimized the PCR cycle number and the template concentration to amplify, and I got acceptable amplicon concentrations (measured by Qubit). The problem starts when I check on a gel or by bioanalyzer I will always have (or almost always) 2 bands/peaks) (1 at 300 bp and other at 400bp). The target amplicon should have about 300 bp so I did a gel purification of the target band but then the concentrations are very low and MiSeq doesn't detect signal and fails to clusterize. I asked BioScientific tech help and they reply it should be from any kind of eukaryotic cells around, but I still think it should not derive in two bands but one.
The options I thought were to prepare a first PCR with universal primers (the same of the kit without illumina adapters) and used as template for the kit library prep, or gel band purification, and I was advice to go with the gel band purification. I did and its always too low yield for Miseq to recognize even with PhiX control added.
So I was wondering if anybody has a clue of what might be happening and any idea how to overpass this. Increasing DNA template concentration is no good since it reaches some kind of limit for the PCR of the kit and does not bring anything new in therms of concentration.
I thank you in advance and sorry for the long text.
Ana
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