SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
MiSeq run with low Q30 and high cluster PF haroldnunez Illumina/Solexa 12 01-06-2017 06:34 AM
Low Cluster Densities in MiSEQ?? Possible explanations??? abyss Illumina/Solexa 30 07-08-2015 10:42 AM
MiSeq v3 2x300 run, Low Q30 ReGenMC Illumina/Solexa 16 02-13-2015 03:51 PM
Newbie question: low cluster density on MiSeq (Chip-Seq pool) HelenaSC Sample Prep / Library Generation 12 07-18-2013 07:28 AM
Run cuffdiff in a computer cluster satp Bioinformatics 1 07-12-2012 11:20 AM

Reply
 
Thread Tools
Old 03-18-2015, 05:06 AM   #1
dross11
Member
 
Location: uk

Join Date: Mar 2015
Posts: 14
Exclamation MiSeq Run with low Cluster PF Help

Hi Everyone I am newbie here and glad I found this wonderful and informative forum. I was wondering if some of you guys can help me out with my MiSeq run stats?

I am using the MiSeq v2 300cycle kits with a targeted sequencing pool of 5 libraries (generated by the Fluidigm Access Array system). I have diluted the pool to 8pM (apparently this gives optimum cluster density with fluidigm libs) and spiked in 5% 12.5pM PhiX. I am also using Fluidigms custom primers.

It is worth mentioning that the kits are out of date by 2 months which may be causing the errors (they were provided to us for free while we are awaiting for new kits to be delivered).

I put the run on 24hrs ago so it is nearing completion and I am getting the following stats:

Cluster density: 988+/-235, Cluster PF: 6.31+/-13.08, Phas/Prephas: 0.288/0.087, Reads: 21.56, Reads PF: 0.93, Q30: 90.8%

The cluster density seems to be just within the optimum range and the Q30 is quite nice but the PF is terrible and I am unsure of why this is the case? Could anyone help me with possible reasons?
dross11 is offline   Reply With Quote
Old 03-18-2015, 05:14 AM   #2
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,787
Default

Best solution in this case is to contact Illumina tech support. They can remotely look at your run and help diagnose.

That said 1000K may be over-clustered for V2 chemistry (especially if these are low nucleotide diversity samples).
GenoMax is offline   Reply With Quote
Old 03-18-2015, 05:19 AM   #3
dross11
Member
 
Location: uk

Join Date: Mar 2015
Posts: 14
Default

I was thinking this, however, I don't believe Illumina provide support for runs that use kits which are out of date. I have noticed people quoting 800K for optimum v2 often for optimum results and 1000K is cutting it quite high.
dross11 is offline   Reply With Quote
Old 03-18-2015, 06:03 AM   #4
lac302
Member
 
Location: DE

Join Date: Dec 2012
Posts: 64
Default

I don't think that's over-clustered enough to produce that low of a % clusters PF. It could be an optics issue. Have a look at the tile images when the run is complete.
lac302 is offline   Reply With Quote
Old 03-18-2015, 06:19 AM   #5
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 498
Default

What percentage of your reads align to phiX? In cases of overclustering, that number is much lower than expected.
HESmith is offline   Reply With Quote
Old 03-18-2015, 06:35 AM   #6
dross11
Member
 
Location: uk

Join Date: Mar 2015
Posts: 14
Default

HESmith - getting 0 but the method on the sample sheet I created is generateFASTQ files only so I'm not sure I should expect much post-analysis other than generating the FASTQ. I'll ask the bioinformatician to have a look at this.

lac302 - They don't seem to be blurry but I am not accustomed to looking at these. I was wondering if someone can have a look at some of the images and confirm? I've attached them to this reply.
Attached Images
File Type: jpg s_1_1101_a.jpg (66.2 KB, 95 views)
File Type: jpg s_1_1107_t.jpg (79.8 KB, 75 views)
File Type: jpg s_1_1109_g.jpg (81.1 KB, 55 views)
File Type: jpg s_1_2102_t.jpg (78.2 KB, 62 views)
dross11 is offline   Reply With Quote
Old 03-19-2015, 04:46 AM   #7
bunce
Member
 
Location: Perth

Join Date: Sep 2012
Posts: 55
Default

Hi Dross11 - your run looks over clustered from the tile images. The thing about over clustering is that the cluster density number (in this case 988k) is somewhat meaningless when the flow cell is this cluttered. We do a lot of V2 Chemistry and try to aim for 700-800K for amplicons. If I were trouble shooting this I would be loading about 30% less... as always it comes down to how you are doing your quant. Cheers Mike
bunce is offline   Reply With Quote
Old 03-19-2015, 05:57 AM   #8
dross11
Member
 
Location: uk

Join Date: Mar 2015
Posts: 14
Default

Thanks for this Mike. I will reduce the conc on my next run and will invest in a kapa lib quant qPCR kit (currently using qubit for quantification).
dross11 is offline   Reply With Quote
Old 03-19-2015, 06:04 AM   #9
bunce
Member
 
Location: Perth

Join Date: Sep 2012
Posts: 55
Default

Hi Dross11 - I think investing in a good qPCR quant is a must if you want to be consistent with your amplicon sequencing. If you want to do it on the cheap we just ordered a synthetic oligo that we titrated into the sweet-spot. Cheers Mike.
bunce is offline   Reply With Quote
Old 03-20-2015, 08:11 AM   #10
esherman
Member
 
Location: USA

Join Date: Jul 2013
Posts: 14
Default

Quote:
Originally Posted by bunce View Post
If you want to do it on the cheap we just ordered a synthetic oligo that we titrated into the sweet-spot. Cheers Mike.
We PCR'd an aliquot of standard from the Kapa qPCR kit for an even cheaper option. We now have 5 liters of standard for less than $20. Buying this from Kapa would cost over $100 million

As far as the low clusters PF problem, we've had MiSeq runs of ultra-low-diversity samples that clustered at 1020K/mm^2 and still had 50% of clusters PF. Having 6% PF with a lower density on a (relatively) more diverse library doesn't sound quite right to me. That said, I would recommend using the qPCR kit for the most accurate quantification, as 988K/mm^2 clusters is admittedly on the high side.

In my opinion, the images look a bit 'grainy', which is typically a result of the MiSeq having to take ultra-long exposures due to low signal intensity. Additionally, a normal MiSeq image will have some clusters that stand out from a noticeably dimmer background (i.e. dye crosstalk). I'm not quite seeing that in all 4 channels. Do you mind sharing the intensity profile and/or signal to noise graphs from Basespace/SAV?
esherman is offline   Reply With Quote
Old 03-20-2015, 08:20 AM   #11
dross11
Member
 
Location: uk

Join Date: Mar 2015
Posts: 14
Default

PCRing the standards is brilliant! I may have to steal this idea :-P

I have attached the requested images, they seemed okay to me and my untrained eye.
Attached Images
File Type: png signal_noise_8A.png (19.1 KB, 56 views)
File Type: png Intensity_chart_8A.png (62.2 KB, 64 views)
dross11 is offline   Reply With Quote
Old 03-20-2015, 08:48 AM   #12
esherman
Member
 
Location: USA

Join Date: Jul 2013
Posts: 14
Default

Quote:
Originally Posted by dross11 View Post
PCRing the standards is brilliant! I may have to steal this idea :-P

I have attached the requested images, they seemed okay to me and my untrained eye.
Thanks for the images!

I'm a bit concerned by the intensities, especially in read 1. Your S/N ratio is surprisingly ok; however, in my talks with Illumina tech support, they say that they never like to see more than one channel with a starting intensity of under 100 (I presume it's in arbitrary fluorescence units?) and it looks like all four of your channels were < 100 for read 1 and half were < 100 for read 2. Since clusters passing filter are determined by the data from the first 25bp of read 1, the chastity algorithm likely had a hard time calling clusters due to the super-low intensities. As a reference, I've included an intensity profile for a sequencing run with custom primers (the big fluctuations are due to alternating high/low diversity regions secondary to our customized homebrew library prep).

My lab had an extremely similar problem (awful intensities in read 1) that was eventually diagnosed as a degraded custom sequencing primer. I assume that you're using Fluidigm's custom sequencing primers? If so, you might want to consider a primer re-synthesis, especially if they're an old (>1 year) synthesis. Other issues may be failure to tap down the cartridge after addition of the custom sequencing primer or a fluidics clog in the read 1 primer line. Illumina tech support can tell you how to perform the fluidics diagnostic procedure.
Attached Images
File Type: png Screenshot 2015-03-20 12.35.52.png (85.7 KB, 54 views)

Last edited by esherman; 03-20-2015 at 08:50 AM.
esherman is offline   Reply With Quote
Old 03-23-2015, 12:45 AM   #13
dross11
Member
 
Location: uk

Join Date: Mar 2015
Posts: 14
Default

esherman - this is extremely helpful, thank you so much.

The fludigm custom sequencing primers are less than a couple months old and have only been used 3 times, so I doubt they are degraded (never say never). Nevertheless, I have ordered in new primers as a precaution and I believe I did tap the cartridge down on the bench after adding the primers but it's difficult to recall such a detail from last week, I will write this down in the protocol to ensure I do this every time without doubt. A fluidic diagnostic test would certainly rule out one factor so I'll see if this can be done.
dross11 is offline   Reply With Quote
Old 03-23-2015, 08:01 AM   #14
esherman
Member
 
Location: USA

Join Date: Jul 2013
Posts: 14
Default

Here are the instructions from an email chain with Illumina tech support detailing how to do a fluidics test on the MiSeq:

Quote:
Go to Manage Instrument – System Check and choose:
Conduct Volume Test
Thermal Ramp Test
Y Stage Test
M3 Mirror Test
Z Stage Test

You can skip the optics tests as they require a special flow cell.

During the Conduct Volume Test, you will need to manually raise and lower the PR2 sipper. Due to this manual movement, the PR2 position may fail the Volume Test without indicating a reagent delivery issue. If a position other than PR2 fails the test, please repeat the test.

Once the tests are complete, you can click on “Show Details” in the lower left of the results screen to get a complete listing of any reagent ports that may have failed.
The volume (fluidics) test takes about an hour or so to complete and requires you to manually raise/lower the PR2 sipper a few times. The rest of the tests only take a few minutes each, so it might not hurt to do them as well, just in case.

I've seen our instrument fail a few of the fluidics tests seemingly at random, only to repeat the test and have all fluidics positions pass. If a position fails, it might not be a bad idea to repeat the fluidics test again. If the same position fails multiple times, then it's probably time to get the instrument serviced.
esherman is offline   Reply With Quote
Reply

Tags
miseq, output, passing filter, v2 300bp

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 01:48 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO