SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
mtDNA sequencing using MiSeq Munch Illumina/Solexa 14 07-26-2018 10:00 AM
miSeq and Exome sequencing isildur Illumina/Solexa 15 03-24-2015 08:37 AM
problem with pooled library cluster generation MiSeq subxero Illumina/Solexa 3 01-08-2014 10:18 AM
problem with R2 reads from Miseq m_elena_bioinfo Bioinformatics 2 04-23-2013 01:41 AM
Covaris parameters to obtain 300~600bp fragments? maureen Sample Prep / Library Generation 0 02-08-2010 09:30 AM

Reply
 
Thread Tools
Old 01-29-2014, 04:39 AM   #1
LizBent
Member
 
Location: Guelph, Ontario, Canada

Join Date: Jan 2012
Posts: 31
Default MiSeq 600bp sequencing problem

Hi, our Genomics Center sent me a link to the MiSeq run they are doing for us with the new 600 bp chemistry - and I notice something on the run stats that scares me:

1. Yield perfect read 1: 170.8M
2. Yield perfect read 4 (second 300 bp read): 9.6M!

The run is still going- will the second read keep going up? It started at 8.9 M for this statistic, and I panicked because in the first attempt to sequence my amplicons, the second read was a lot lower than the first.

I'm worried because I added indexes and adapters via PCR (regular primer, then adapters+ regular primers, then indexes+adapters in 3 sequential PCRs) and I'm so afraid I screwed that up.


Last edited by LizBent; 01-29-2014 at 04:43 AM. Reason: wrong info for second read
LizBent is offline   Reply With Quote
Old 01-29-2014, 05:17 AM   #2
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

Quote:
Originally Posted by LizBent View Post
Hi, our Genomics Center sent me a link to the MiSeq run they are doing for us with the new 600 bp chemistry - and I notice something on the run stats that scares me:

1. Yield perfect read 1: 170.8M
2. Yield perfect read 4 (second 300 bp read): 9.6M!

The run is still going- will the second read keep going up? It started at 8.9 M for this statistic, and I panicked because in the first attempt to sequence my amplicons, the second read was a lot lower than the first.

I'm worried because I added indexes and adapters via PCR (regular primer, then adapters+ regular primers, then indexes+adapters in 3 sequential PCRs) and I'm so afraid I screwed that up.

The indexes and adapter sequences should be on the same oligo, so if you messed up the indexing PCR, you'd fail to cluster. What's your cluster density?
TonyBrooks is offline   Reply With Quote
Old 01-29-2014, 05:38 AM   #3
LizBent
Member
 
Location: Guelph, Ontario, Canada

Join Date: Jan 2012
Posts: 31
Default

Quote:
Originally Posted by tonybrooks View Post
the indexes and adapter sequences should be on the same oligo, so if you messed up the indexing pcr, you'd fail to cluster. What's your cluster density?
1067 +/- 23
LizBent is offline   Reply With Quote
Old 01-29-2014, 05:45 AM   #4
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,978
Default

Is this a 2D barcode or 1D barcode run? V2 or V3 chemistry?

If this is V2 chemistry and these are amplicons then this run is overloaded at 1000+ clusters/mm^2.

Are you sure you have 170M reads for Read 1?
GenoMax is offline   Reply With Quote
Old 01-29-2014, 05:52 AM   #5
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

Optimal loading for amplicon on V2 is around 800K/mm2, but it's very library specific.
We've had libraries run at 1000k/mm2 with no problem at all.

Either way, it looks like you library is clustering. Next thing to check is the Phas/Prephas. You're looking for values below 0.1% for your reads, indexes are usually higher. Higher values indicate a possible problem with your reagents.

The % perfect applies only to the PhiX spiked into the run, hence the low yield.
TonyBrooks is offline   Reply With Quote
Old 01-29-2014, 07:30 AM   #6
AllSeq
Registered Vendor
 
Location: San Diego, CA

Join Date: Oct 2013
Posts: 138
Default

Quote:
Originally Posted by GenoMax View Post
Are you sure you have 170M reads for Read 1?
Same question: are you sure you have 170M reads? The spec is 25M.
__________________
AllSeq - The Sequencing Marketplace
info@AllSeq.com
www.AllSeq.com
AllSeq is offline   Reply With Quote
Old 01-29-2014, 07:58 AM   #7
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

Quote:
Originally Posted by AllSeq View Post
Same question: are you sure you have 170M reads? The spec is 25M.
Pretty sure you're looking at the "Yield Perfect" column in BaseSpace, which is the PhiX reads. M = Mb here, not number of reads
My last run had 12.78M reads pass filter = 3.2Gb for R1 (250bp * 12.78M)
I has 10% PhiX spiked in, so 320Mb of that 3.2Gb was PhiX
Yield Perfect was 240M, so I had ~75% %Pefect (in actual fact, it was 75.4% according to BaseSpace)
TonyBrooks is offline   Reply With Quote
Old 01-29-2014, 04:24 PM   #8
LizBent
Member
 
Location: Guelph, Ontario, Canada

Join Date: Jan 2012
Posts: 31
Default

The run statistics are getting better, so I think I was freaking out for no reason... I have 3.4G of data from read 1, and it's halfway through read 2 and there are 1.7G of data, so this run should (?) be fine...

Sorry to have bothered you all. I really hate the BaseSpace run summary interface!
LizBent is offline   Reply With Quote
Old 01-29-2014, 04:35 PM   #9
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,978
Default

At times like these having less (or not continuously updated) information actually turns out to be better :-)
GenoMax is offline   Reply With Quote
Old 01-30-2014, 05:53 AM   #10
microgirl123
Senior Member
 
Location: New England

Join Date: Jun 2012
Posts: 197
Default

Quote:
At times like these having less (or not continuously updated) information actually turns out to be better :-)
So true. I just started my first V3 run Tuesday so I was watching it closely on Basespace. First up pops the cluster density at 523 +/- 477! Panic inducing! Turns out to be 983, which is perfect. Then clusters passing filter popped up at 43% +/- 52%! Completely useless information but worrisome. Turns out to be 91%, so I'm happy.
microgirl123 is offline   Reply With Quote
Reply

Tags
illumina, miseq, quality control

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 10:24 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO