AmpliSeq Cancer Panel

[SarahNGS]

Does anyone here have experience with the Ion Torrent Ampliseq Cancer Panel? I have a couple of questions-
How satisfied are you with the results?
How many samples are you running per batch?

[NextGenSeb]

Hi Sarah,
I have had a couple of runs on the v1 ampliseq cancer panel, with mixed results re: amplicon coverage. Some appear to drop out systematically, however I understand that is a well known issue with the panel. The new v2 panel is supposed to fix that, but I have just started testing that so can't comment there. As for the data quality, at this stage it seems to be lower than what you get of a MiSeq or related system, especially single base deletions seem overly frequent. But it is a lot faster and at this stage at least the most widely available option.

As for the samples, I am using 314 chips, hence only one sample per run. There is the option to run more on a bigger chip though.

Cheers
Seb

[[email protected]]

I have ran 3 indexed samples in a 316 chip and 6-8 indexed samples in a 318 chip. The coverage has increased significantly after conversion to 200bp chemistry but in my opinion there are still many variables that can lead to read quality (not to mention the intrinsic variability of coverage in the v1 panel). There are several systemic, recurrent calls that I notice (JAK - since corrected, APC...). We have used FFPE derived DNA (some as old as 20 yr), cell line derived DNA, and Fz tumour DNA in the AmpliSeq cancer panel with satisfactory results. Workflow is pretty fast - from DNA to results in 2 days.

Illumina has an analogous solution in TruSeq - love to see how it compare (am sure will get higher read depth and less inter-operator variability).

Stephen

[NextGenSeb]

Hi all,

have to agree with Stephen, the v2 panel appears to be better than v1, both in coverage and workflow. But I do get those APC calls as well.

@ Stephen
Can you please elaborate on how you determined those calls are systemic errors and which other ones you see?

Cheers
Seb

[snetmcom]

Hi all,

have to agree with Stephen, the v2 panel appears to be better than v1, both in coverage and workflow. But I do get those APC calls as well.

@ Stephen
Can you please elaborate on how you determined those calls are systemic errors and which other ones you see?

Cheers
Seb

Ion has stated v1 has systemic issues with JAK2. The current bed files are clearly marked , NO JAK2.

[slm1816]

Little experience with both cancer panels

Hi,

I have run the Ion Torrent AmpliSeq Panel and the Illumina TruSeq Panel.

Starting with the Illumina TruSeq Panel:
-Much easier setup. The machine might run longer but the ease of setup and the "walk away" time is much more. It allows you to walk away and complete other experiements you are working on.

I have run 3 samples with barcoding so far. I have plans to do a much larger batch soon. I got satisfactory results.

-After the run is complete, the MiSeq reporter does all the demultiplexing, aligment, etc which is wonderful! You can view results in MiSeq Reporter and Amplicon Viewer. There are great tools from Illumina, however, they are not useful in catching indels. They are great for point mutations. I am still trying to find a good third party software that will catch indels. Any suggestions?

-coverage is much better with the Illumina Platform.
-Working on adjusting the input for better cluster densities.
-Having issues with the company is getting answers to some of my questions and some of the excel sheets they have given me, such as the list of genes and the exons covered have been wrong. Just have to scrutinize everything

Ion Torrent Ampliseq:

-Lots of hands on time.
-have had major problems with initializing the machine
-Only run one sample at a time on a 316 chip.
-Ion Reporter is similar to the MiSeq reporter but some of the columns of data output are wrong. For instance, a G12D mutation of the KRAS gene is not on exon 3. That's what the reporter is telling us. So it looks like you have to scrutinize all of that as well.

That's all I can think of off the top of my head right now.


This is all such a major experiement. I learn something new everyday and sometimes it gets very frustrating!
I would appreciate it if we can all help each other out!

[IonTorrent]

Hi slm1816,

Thanks for reporting the issue with the KRAS G12D exon number in Ion Reporter™ Software. We confirmed this error recently and have identified a solution. You can expect an update early next week that will resolve this issue; we'll send out an announcement to users when the update is complete. Please let us know if we can help further.

Also worth pointing out is that if you ever have an urgent question or suspect an issue I'd recommend the following paths: 1) as a US customer, for fastest response call 1-87-SEQUENCE to reach one of our Technical Applications Scientists [other regions have their own customer support phone numbers], or 2) contact your local Field Applications Scientist (who I understand is already working with you on the initialization issues you reported here and in another thread). For less urgent matters, or general inquiries, consider the Ion Community.

Hi,

I have run the Ion Torrent AmpliSeq Panel and the Illumina TruSeq Panel.

Starting with the Illumina TruSeq Panel:
-Much easier setup. The machine might run longer but the ease of setup and the "walk away" time is much more. It allows you to walk away and complete other experiements you are working on.

I have run 3 samples with barcoding so far. I have plans to do a much larger batch soon. I got satisfactory results.

-After the run is complete, the MiSeq reporter does all the demultiplexing, aligment, etc which is wonderful! You can view results in MiSeq Reporter and Amplicon Viewer. There are great tools from Illumina, however, they are not useful in catching indels. They are great for point mutations. I am still trying to find a good third party software that will catch indels. Any suggestions?

-coverage is much better with the Illumina Platform.
-Working on adjusting the input for better cluster densities.
-Having issues with the company is getting answers to some of my questions and some of the excel sheets they have given me, such as the list of genes and the exons covered have been wrong. Just have to scrutinize everything

Ion Torrent Ampliseq:

-Lots of hands on time.
-have had major problems with initializing the machine
-Only run one sample at a time on a 316 chip.
-Ion Reporter is similar to the MiSeq reporter but some of the columns of data output are wrong. For instance, a G12D mutation of the KRAS gene is not on exon 3. That's what the reporter is telling us. So it looks like you have to scrutinize all of that as well.

That's all I can think of off the top of my head right now.


This is all such a major experiement. I learn something new everyday and sometimes it gets very frustrating!
I would appreciate it if we can all help each other out!

[Nitrogen-DNE-sulfer]

Has anyone tried to move the Ampliseq to MiSeqs? 48 genes for TruSeq seems a bit limiting.

[DNADEB]

Does anyone know of any publications using Ampliseq panels with Ion PGM?

[[email protected]]

We had a paper in J Neurosurgery Pediatrics (2012 9;517-23) in which we used the cancer panel on primary and recurrent tumour from the same patient. It was pretty rough and in the early phase of assay development and we didn't have access to blood. We have submitted an abstract to ASCO GI to discuss the results of profiling 200 cases of FFPE gastric cancers using the panel. Also, we have several other manuscripts in different stages of development which used the cancer panel as well as customized panels for SEQVAL.

[DNADEB]

Thanks for your reply!