We generated PCR products of different lenghts ( 200 and 2,000 nts ) from different types of assays: RACE-PCR, cDNA library inserts, amplicons from mutated DNA sites in tumors with heterogeneous population mixes of normal and tumor cells. We need to (1) obtain the heterogeneous sequence reads present in these PCR products and (2) get the relative copy number present in the samples.
Question 1. What technology is recommended ? What library generation (principle / paper / kit ) ?
Question 2. Where can I get those PCR products sequenced - after appropriate preparation ?
Thank you
Anton Wellstein, MD PhD; Professor Georgetown University, Lombardi Cancer Center, Washington DC e-mail: [email protected]
Dear Anton,
(1) We have been working on deep sequencing of amplicons using Roche 454 and IonTorrent. The typical workflow was generation of the amplicons, attachment of adapter sequences together with an 8 bp multiplex barcode at the end where you start sequencing (because sequence quality is optimal there) in a nested PCR setup, followed by cleanup of these amplicons using Qiagen's QIAquick PCR Purification kit, but other will likely work as well.
We then used the deep seq kits that the manufacturer recommends.
If your amplicons have different sizes, I quantification gets all the more difficult. These methods are often used in gene therapy settings to evaluate outgrowth of clones. In the paper below, we carefully looked how good reads and spike in or qPCR measurements of clones correlate, which wasn't very good and quite variable. Sticking a DNA barcode into your products and limiting the PCR cycles before you sequence might help improve quantification if such changes are at all possible in your method.
(2) I have previously used gatc-biotech.de in Germany for deep sequencing, which worked out nicely, but there are probably plenty of small biotech companies that will do it for a reasonable price. Try to go for one that has experience in sequencing amplicons. They will probably be able to help you troubleshoot a bit here and there.
For your mutation screen you might want to have a look at the Thol et al manuscript linked below.
Hope this helps.
Article Evaluating a Ligation-Mediated PCR and Pyrosequencing Method...
Article Next-generation sequencing for minimal residual disease moni...
Dear Anton,
(1) We have been working on deep sequencing of amplicons using Roche 454 and IonTorrent. The typical workflow was generation of the amplicons, attachment of adapter sequences together with an 8 bp multiplex barcode at the end where you start sequencing (because sequence quality is optimal there) in a nested PCR setup, followed by cleanup of these amplicons using Qiagen's QIAquick PCR Purification kit, but other will likely work as well.
We then used the deep seq kits that the manufacturer recommends.
If your amplicons have different sizes, I quantification gets all the more difficult. These methods are often used in gene therapy settings to evaluate outgrowth of clones. In the paper below, we carefully looked how good reads and spike in or qPCR measurements of clones correlate, which wasn't very good and quite variable. Sticking a DNA barcode into your products and limiting the PCR cycles before you sequence might help improve quantification if such changes are at all possible in your method.
(2) I have previously used gatc-biotech.de in Germany for deep sequencing, which worked out nicely, but there are probably plenty of small biotech companies that will do it for a reasonable price. Try to go for one that has experience in sequencing amplicons. They will probably be able to help you troubleshoot a bit here and there.
For your mutation screen you might want to have a look at the Thol et al manuscript linked below.
Hope this helps.
Article Evaluating a Ligation-Mediated PCR and Pyrosequencing Method...
Article Next-generation sequencing for minimal residual disease moni...
Dear Dr. Anton, I too agree with Dr. Martijn Brugman.
But as per your query related with copy number and heterogenous sequence identification,
Kindly use paired end sequencing like Illumina HIseq. with higher coverage will be the good way to get optimal result.
Library prep is going to be a challenge with your larger fragments. You could potentially use the HiSeq or MiSeq but again, making a library might pose an issue in terms of cluster generation and your sequencing data getting swamped by the smaller amplicons. I'd try to minimally get my sequencing library to be similar in length either through size selection methods . Ion would be a good alternative but typically your amplicons need to be about 100-400bp (depending on the kit) in order to have efficient ion sphere templating. Dosage or CNVs in samples can be determined by including a sample barcode in your PCR primer
We are using miSeq (250 cycle paired-end) with Nextera sample prep to quantify T-cell clones in 5'RACE amplicons of TCRs. It is amazingly quantitative, as proven by extensive dilution-curve spiking with RNA from monoclonal T-cell leukemia. We have a paper in the peer-review system I would be glad to share.
Thank you so much for the answers. Very helpful - we have access to a MiiSeq in our core and this looks like a feasible approach we will take initially.
Constantin, if you would not mind sending me a pdf of your paper, I would very much appreciate that. My e-mail is [email protected]
Best
Anton
We are using MiSeq (250 cycle paired-end) with Nextera XT sample prep. kit for PCR, longPCR, specific cDNA too.
Nextera XT has one limit, DNA must be > 300pb. You need 1ng od PCR product only.
We have 100% conformity with Sanger.
Josef
We are also using the Miseq technology in our lab, but with the 300 bp paired-end and it's working quit fine. I think with this technique you can look to fragments with sizes till 500bp (If I were you I wouldn't go any higher so you still have a good overlap + the quality of the reads drops below 20 after 250bp)
The Nextera XT library prep was also for me a good start and I really recommend it. I attached the protocol Illumina provided last December.
Good luck with it!
Caroline
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