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Old 11-08-2011, 10:17 AM   #1
csquared
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Default Bottom Middle Swath and other focus issues

Is anyone else seeing a pretty frequent loss of lanes or substantially reduced lane output due to focus issues related to bubbles in lanes or other focus issues when using v3 chemistry on the HiSeq?

In our case, there is an instrument dependency where some HiSeqs are better than others. I know Illumina is working towards a fix for this problem but I was hoping to get an idea if the frequency we are seeing this issue is unusual.

On the worst instrument, we are seeing it in the majority of lanes, losing anything from 30-60% of the lane output. The data can be cleaned up, but cleaning things up on a tile-by-tile basis becomes more time consuming than it is worth. Best machines we see it for a cycle or two in a single lane or so with minimal, but measurable effect in the final data.

Hoping to hear what others are seeing. Thanks.
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Old 11-09-2011, 05:54 AM   #2
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Yep, it's affecting a large swath (lol) of us:
http://seqanswers.com/forums/showthread.php?t=13830
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Old 11-09-2011, 06:58 AM   #3
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We got a message from illumina regarding this quite recently (see attachment)
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Old 02-28-2012, 05:53 AM   #4
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Question Loss of focus and data

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Originally Posted by RPiddock View Post
We got a message from illumina regarding this quite recently (see attachment)
As far as we know, there has been no solution offered for the problem of bubbles/focus/BMS dropout yet. Anyone heard otherwise?
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Old 02-28-2012, 05:58 AM   #5
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No solution has been given to us I'm afraid.
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Old 02-28-2012, 07:27 AM   #6
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The latest HCS (1.5.1) should fix it:

Quote:
Originally Posted by Illumina
Bottom Middle Swath Update
Jan 18 2012
Occasionally, a HiSeq will be unable to correctly focus on a swath (1/6th of a lane on the flowcell) for a single cycle. This usually happens on the middle swath of the bottom surface of the flow cell, hence the name BMS (for "bottom middle swath"). Illumina has determined that when a bubble forms at the inlet of a lane, it can prevent accurate determination of the focus position needed to scan a swath from that lane. As a result, the data from that swath is sometimes out of focus and could result in no-call bases ("N" basecalls) for just that cycle. The wider lanes on the v3 flowcells cause this loss of focus most often on the bottom middle swath.

Illumina R&D has developed a new version of the HiSeq firmware that reduces occurrences of BMS. This works by identifying bubbles in the region of the lane used for focusing and preventing focusing on bubbles. During testing of this firmware, they identified some changes that would lead to better identification and avoidance of bubbles. These changes were made in a new firmware version, which is currently undergoing final testing. We expect to release this firmware in February 2012.

If you are experiencing occasional loss of focus resulting in a drop in intensity and base calls of "N" for the affected cycle, be sure you have upgraded to HCS 1.4.8 as a mitigation until the new firmware is released . If you have additional questions, please contact Illumina Technical Support at techsupport@illumina.com.
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Old 02-28-2012, 09:36 AM   #7
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Quote:
Originally Posted by GW_OK View Post
The latest HCS (1.5.1) should fix it:
If it's ever available.

"Thank you for contacting Illumina Technical Support.

We are still completing testing for this "fix". We donít have an exact timeline at the moment, but we will let you know when the information is available. Thank you for your patience.

If you have any additional questions, please feel free to email or call us.

Kind regards,
Zheng"

Zheng Xu, Ph.D.
Technical Applications Scientist
Illumina Technical Support
Illumina, Inc.
North America: 800.809.4566
Global contacts: www.illumina.com/support.ilmn
Website: www.illumina.com
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Old 02-28-2012, 10:47 AM   #8
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But it is available? We've got it. It's on icom:

Quote:
Originally Posted by icom
HCS 1.5.15 is released for HiSeq and HiScan SQ

Feb 21 2012

HCS 1.5.15 is now available for download to your HiSeq and HiScan SQ systems. New features include support for dual indexing and warnings when there is not enough disk space available to start the sequencing run. HCS 1.5.15 also fixes a memory leak that could lead to the RTA software freezing up, and has a new tile layout that reduces the RTA memory footprint. Release notes and the installer can be found at https://my.illumina.com/download/sum...9UKStokGqhI96Q.

For further assistance please contact techsupport@illumina.com.
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Old 02-28-2012, 11:23 AM   #9
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Default not the 'fix'

The release notes say nothing about "BMS" or bubble problem.
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Old 02-28-2012, 12:49 PM   #10
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Nope, 1.15.1 doesn't fix the BMS, supposedly the next release - but have heard that before.
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Old 02-28-2012, 01:26 PM   #11
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Huh. Well.
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Old 02-28-2012, 04:39 PM   #12
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Quote:
Originally Posted by GW_OK View Post
Huh. Well.
So does this mean you haven't been adversely affected by loss of focus?
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Old 02-29-2012, 09:39 AM   #13
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I have had BMS dropouts, yes, but there seems to be less of it now, if any. Whether it's the new HCS, or the fiddling (something about changing the focus window) my FES did when he was here on an unrelated matter I can't say for sure.
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Old 03-01-2012, 06:21 AM   #14
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We are still seeing LOTS of it, and engineers have been out here twice, disassembled the instrument and put it back together. No use. See the pretty picture. I can find lots more. Also note that this isn't bottom middle, it's bottom left and bigger than a single swath anyway. I checked our software and we're at the latest version (engineers did the installation). Guess your engineers are better fiddlers or we've got a lemon.
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Old 03-01-2012, 07:15 AM   #15
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You picture is a bubble, it looks like, not exactly killing your entire swath. I've got bubbles like that for sure, but it's not the dreaded BM(LR)S.
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Old 03-01-2012, 07:20 AM   #16
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Default Bubbles not BMS

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Originally Posted by GW_OK View Post
You picture is a bubble, it looks like, not exactly killing your entire swath. I've got bubbles like that for sure, but it's not the dreaded BM(LR)S.
Okay, I'm conflating the two problems, sorry. I should call it something else. But they're frequent, big, and move during the run so we lose lots of data. What *is* the solution? Apparently degassing reagents is not advised.
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Old 03-01-2012, 08:26 AM   #17
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Default Swath dropout

Here's the swath dropout picture. Top middle. Also occurred on bottom middle.
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Old 03-01-2012, 10:58 AM   #18
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Quote:
Originally Posted by HMorrison View Post
Here's the swath dropout picture. Top middle. Also occurred on bottom middle.
Yep, that's a swath loss. And I did try degassing the scan mix once but it didn't seem to do anything to lessen the bubbles. The bubbles aren't the end of the world, though, at worst you'll have an N in your sequence. And since degassing didn't seem to do much I'm at a bit of a loss as to how to combat it as well.
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Old 03-01-2012, 11:03 AM   #19
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Current run...top middle and bottom middle swath dropout on lane 4 at cycles 17, 38, 40, 52, 57, 58 and counting. Haven't checked other lanes. Ns in sequences hurt; we are doing 16s tag analysis as well as genomic/metagenomic libraries.
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Old 03-01-2012, 07:04 PM   #20
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Dang, that is a ton of dropouts. I hope Illumina is comp-ing you kits.
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