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Forum: Ion Torrent 01-06-2014, 12:46 AM
Replies: 5
Views: 3,282
Posted By JPC
qubit for quant, but we also use the bioanalyser...

qubit for quant, but we also use the bioanalyser tp make sure the profile looks as expected (no primer etc.)

JPC
Forum: Ion Torrent 01-03-2014, 03:15 AM
Replies: 5
Views: 3,282
Posted By JPC
We run a Proton and found that we improved our...

We run a Proton and found that we improved our quality by reducing the quantity of DNA that goes into the emPCR by by 20%, we now do this is standard

JPC
Forum: Bioinformatics 10-28-2013, 01:56 AM
Replies: 0
Views: 1,039
Posted By JPC
make: *** [htscmd] Error 1

Hello All, I'm trying to build GATK on my iMac (OS X 10.9 Mavericks) but when installing htmlib I get the following;

vcfcheck.c:703:39: warning: format specifies type 'long' but the argument has...
Forum: Ion Torrent 09-13-2013, 08:17 AM
Replies: 8
Views: 8,286
Posted By JPC
In case anyone else is interested I downloaded...

In case anyone else is interested I downloaded some exome data from Ion Community and converted thee BAM files to put them into fastQC, the results are very similar to what we are seeing with Q20 to...
Forum: Ion Torrent 09-10-2013, 01:41 AM
Replies: 8
Views: 8,286
Posted By JPC
Question Ion Proton Q20

Hello All, we've only just done our first Proton run and the per base quality of the FastQC has ~90% of our data in the Q20 to Q28 range, should I be able to get up into the 30s or is it normal to be...
Forum: SOLiD 09-04-2013, 08:43 AM
Replies: 1
Views: 1,997
Posted By JPC
We only use E120s but in order to get passed 200k...

We only use E120s but in order to get passed 200k it is just a case of starting out with more beads, how may beads are you trying to put down each lane?

JC
Forum: Illumina/Solexa 09-04-2013, 08:40 AM
Replies: 8
Views: 5,900
Posted By JPC
We've pooled PCR products for the MiSeq but had a...

We've pooled PCR products for the MiSeq but had a terrible time getting them balanced, equimolar pooling was way off, it took a bit of 'trial and error' to get to an acceptable level

JPC
Forum: Ion Torrent 07-30-2013, 03:03 AM
Replies: 2
Views: 4,272
Posted By JPC
Hi vandy, I assume you've moved on from this now,...

Hi vandy, I assume you've moved on from this now, did you try and save the reaction? in my experience with Sureselect this step is extremely sensitive so we would have started again if this had...
Forum: SOLiD 07-10-2013, 04:06 AM
Replies: 5
Views: 3,688
Posted By JPC
If you have 25% mapping and 75% off target then...

If you have 25% mapping and 75% off target then either your hyb failed of your post hyb washing wasn't efficient. I wouldn't suspect FFPE at this stage if the only issue with your data is the off...
Forum: SOLiD 06-10-2013, 08:07 AM
Replies: 1
Views: 2,605
Posted By JPC
RNA has to be reverse transcribed (turned into...

RNA has to be reverse transcribed (turned into DNA) before it can be sequenced, even if there was RNA in your sample it would not be present in your data
Forum: Illumina/Solexa 05-24-2013, 12:18 AM
Replies: 8
Views: 2,380
Posted By JPC
I'm not sure when they were added as this is the...

I'm not sure when they were added as this is the first protocol I have used on the MiSeq

-Nextera XT Sample Preparation Guide 15031942. Pages 30/31
Forum: Illumina/Solexa 05-23-2013, 07:51 AM
Replies: 8
Views: 2,380
Posted By JPC
Hi, the Nextera_XT kit already has Ampure beads...

Hi, the Nextera_XT kit already has Ampure beads for the clean up, I've used this on the MiSeq with no problems.

JPC
Forum: Illumina/Solexa 05-16-2013, 07:50 AM
Replies: 7
Views: 2,351
Posted By JPC
Yes we have tried tagmentation and confirmed the...

Yes we have tried tagmentation and confirmed the sequences with Sanger, also there is no correlation with product size and success (nor GC content, or even % of repetitive DNA).

While I do...
Forum: General 05-15-2013, 08:37 AM
Replies: 26
Views: 3,075
Posted By JPC
You are vastly underestimating the complexity of...

You are vastly underestimating the complexity of the human body, there are 90 trillion cells in each of us each and they are in a constant state of flux, each cell has 3 billion bases of DNA, also...
Forum: Illumina/Solexa 05-15-2013, 08:03 AM
Replies: 7
Views: 2,351
Posted By JPC
Hi microgirl, we are pooling the PCR then...

Hi microgirl, we are pooling the PCR then prepping it as one library so there are no adapters to check for at the pooling stage. When we pool 2 libraries we get good balance between the samples but...
Forum: General 05-15-2013, 07:54 AM
Replies: 26
Views: 3,075
Posted By JPC
In the most simple terms you are saying "if you...

In the most simple terms you are saying "if you can detect the cells and kill them you would have a cure" then 'Yes' that is true but that does not take into account the complexity of the biological...
Forum: General 05-15-2013, 06:25 AM
Replies: 26
Views: 3,075
Posted By JPC
It's hard to explain why the answer to this is...

It's hard to explain why the answer to this is 'No' as I'm guessing you don't have much of a background in genetics so I'll try a computer based analogy.

Your anti virus software can identify and...
Forum: Illumina/Solexa 05-15-2013, 12:35 AM
Replies: 7
Views: 2,351
Posted By JPC
We shear with the Covaris and check it with the...

We shear with the Covaris and check it with the Bioanalyser, we've also checked with agarose gel to see if there is a significant quantity of un-sheared product, on top of that we've seen the same...
Forum: Illumina/Solexa 05-14-2013, 08:40 AM
Replies: 7
Views: 2,351
Posted By JPC
pooling long range PCR

Hello, I have an experiment with 20 over lapping long range PCR fragments that span a single gene. The average size is 5kb (max 10, min 1.5kb) with equimolar pooling the balance between fragments is...
Forum: SOLiD 05-01-2013, 04:38 AM
Replies: 25
Views: 9,164
Posted By JPC
Ah sorry, getting my threads mixed up!

Ah sorry, getting my threads mixed up!
Forum: SOLiD 05-01-2013, 03:08 AM
Replies: 2
Views: 923
Posted By JPC
We've done something similar with Yeast (spiking...

We've done something similar with Yeast (spiking it into an E120)

No the low % of the 3 virus barcodes does not cause you trouble
Yes the colour balance should be unaffected
We don't see the...
Forum: SOLiD 05-01-2013, 03:05 AM
Replies: 2
Views: 1,084
Posted By JPC
Hi jayalakshmi we've seen this problem...

Hi jayalakshmi

we've seen this problem before and traced the issue back to a problem with quantification, what method do you use?

Too much input DNA leads to polyclonal beads on the slide which...
Forum: SOLiD 04-29-2013, 04:32 AM
Replies: 6
Views: 1,486
Posted By JPC
ah I see, we're still waiting for installation of...

ah I see, we're still waiting for installation of our Ion, I had assumed it would still require the Covaris, it would be nice if someone at LifeTech had mentioned a new shearing technique to me!
Forum: SOLiD 04-29-2013, 04:28 AM
Replies: 25
Views: 9,164
Posted By JPC
It's a little lower than you might like but we've...

It's a little lower than you might like but we've seen runs like this. It equates to around 150,000 reads per panel doesn't it?
Forum: SOLiD 04-26-2013, 08:23 AM
Replies: 3
Views: 3,087
Posted By JPC
With Solid data you need to do the mapping in...

With Solid data you need to do the mapping in base-space, not convert the fastq and map that. If you're not familiar with it I would recommend tracking down an expert.

I don't know if it is still...
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