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Old 09-21-2012, 02:03 PM   #21
ECO
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Quote:
Originally Posted by TonyBrooks View Post
We're getting similar problems, compounded by low diversity.
Our library is an pool of three different amplicons with three different R1 sequencing primers. We added some N's in our amplicon generation primers to immediately follow R1 primer annealing site in order to increase diversity during cluster registration. Apparently, it wasn't enough as our run was terrible (160k/mm2 density - Q-scores along the floor). We're going to repeat with 5% PhiX and the Josh Quick/Nick Loman fix (http://pathogenomics.bham.ac.uk/blog...illumina-miseq)

The issue I have is that several things worry me when looking at the thumbnails and general SAV analysis

1) Density is clearly higher than 160k/mm2 when eyeballing the thumbnails (poor cluster registration due to low diversity?)
2) There are a mix of low and high intensity clusters that can't be explained by laser cross-talk. These low diversity clusters are appearing on all channels (ATCG) not just the alternate base from the respective laser which is a bit strange, no? You can clearly see the same general cluster pattern for all bases - as if there's cross talk between all bases with some clusters lighting up fairly bright.
3) Only one amplicon seemed to generate any data post indexing

I'm wondering if two of the three read primers have too low Tm and hence generating lower signal. These are being filtered out leading to the lower cluster density estimate.
Tm's of each of the R1 primers are ~66 according to OligoCalc basic. Maybe I need to add some bases on to the other oligos? How do people check their Tm's? MiSeq runs at 65, if I'm not mistaken.

Comments and theories welcome!
The MiSeq Recipe for priming R1 is the following:

Code:
<ChemistryStep Description="FirstReadPreparation">
	<PumpToFlowcell ReagentName="LDR" AspirationRate="250" DispenseRate="2500" Volume="75" />
        <Temp Temperature="65" Duration="60000" />
        <PumpToFlowcell ReagentName="ReadOnePrimer" AspirationRate="2000" DispenseRate="2500" Volume="300" />
        <PumpToFlowcell ReagentName="ReadOnePrimer" AspirationRate="250" DispenseRate="2500" Volume="75" />
        <Temp Temperature="40" Duration="60000" />
        <PumpToFlowcell ReagentName="PR2" AspirationRate="2000" DispenseRate="2500" Volume="120" />
        <TempOff />
    </ChemistryStep>
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Old 09-23-2012, 03:31 AM   #22
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Hi Tony,

1) Agreed it looks about 2x that. lower reported density than you see in the Thumbnails could be caused by registration failures, you should see 0's in the P90 A/C/G/T if there was a registration failure in the imaging tab on SAV.
2) You expect to see some cross talk in A/C/T but G is clean, in your images G still has crosstalk which does suggest a mixed signal in the cluster - this would also result in a very low %PF which you see as it is a signal purity filter. Not sure what would cause this... Can you post the average matrix for the lane from Data>Intensities>Basecalls>Matrix>s_1_matrix.txt, it might give some clues.

The Tm's of the primers should be ok judging by your intensity plot (from @jamimmunology). If they are too low the intensity bombs between cycle 1 and 2 - your intensity actually looks quite good initially (C=1600) but something very strange happens around cycle 100.

Josh
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Old 10-29-2012, 04:38 PM   #23
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We encountered similar problem before (clusters on all channels) when we used sequencing primers annealing to P5/P7. Then we fixed the issue by using primers with similar Tm but annealing to other parts. What sequencing primers are you using?

Quote:
Originally Posted by TonyBrooks View Post
We're getting similar problems, compounded by low diversity.
Our library is an pool of three different amplicons with three different R1 sequencing primers. We added some N's in our amplicon generation primers to immediately follow R1 primer annealing site in order to increase diversity during cluster registration. Apparently, it wasn't enough as our run was terrible (160k/mm2 density - Q-scores along the floor). We're going to repeat with 5% PhiX and the Josh Quick/Nick Loman fix (http://pathogenomics.bham.ac.uk/blog...illumina-miseq)

The issue I have is that several things worry me when looking at the thumbnails and general SAV analysis

1) Density is clearly higher than 160k/mm2 when eyeballing the thumbnails (poor cluster registration due to low diversity?)
2) There are a mix of low and high intensity clusters that can't be explained by laser cross-talk. These low diversity clusters are appearing on all channels (ATCG) not just the alternate base from the respective laser which is a bit strange, no? You can clearly see the same general cluster pattern for all bases - as if there's cross talk between all bases with some clusters lighting up fairly bright.
3) Only one amplicon seemed to generate any data post indexing

I'm wondering if two of the three read primers have too low Tm and hence generating lower signal. These are being filtered out leading to the lower cluster density estimate.
Tm's of each of the R1 primers are ~66 according to OligoCalc basic. Maybe I need to add some bases on to the other oligos? How do people check their Tm's? MiSeq runs at 65, if I'm not mistaken.

Comments and theories welcome!
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Old 10-30-2012, 01:49 AM   #24
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The sequencing primers are those used in the PCR for one end of the amplicons (25 or 26 nt), plus a different number of P5 nucleotides (either 6 or 9, depending on the primer) to bring the Tms in line with SP1.

My thought was that even if there is binding of sequencing primers to the lawn via their 5's, surely that shouldn't lead to fluorescence, as there's a 25/6 nt 3' overhang, so nothing for polymerase to act on? Although I can see how this could reduce the effective concentration of primer in the mix.

We actually can't move the primer any further into the amplicon, due to the variable nature of what we're sequencing, although I'm now toying with using LNA modifications to bring the Tms in line/above the 65C mark without increasing (or maybe even reducing) the number of P5 nucleotides used. (Anyone have any recommendations for LNA sequencing primer design?)
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Old 02-11-2014, 06:16 AM   #25
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We experience a problem with MiSeq in cluster generation (or cluster detection). We sequence PCR product of a certain gene and the P5 and P7 sequences for the adaptors and dual-indices are added during PCR (pair end).
We pool approx. 350 samples in subpools (with equal conc. of each sample), run on the gel, gel purify, quantify once again and form final pool (with equal conc. of subpools). The final pool quality and quantity looks perfect (for the last run we have purified even twice: 1. gel purification; 2. AMPure PCR purification). The subpools and final pool quantity has been estimated with Agilent 7500 DNA Chip. The first time we loaded on MiSeq 12pMol and the number of clusters formed was 188 with an output of 0.5million reads. We have rerun the same library loading 60pMol, which resulted in 1200 clusters and 5.2G sequences.
Illumina support refuses to help us, as it is self-designed primers.
We have reconsidered all the possible and impossible reasons and looked library preparation and quantification protocols numerous times. So I decided to inquire here, maybe somebody has experienced similar problems or knows what could be the possible explanation for this? Help or insight is really appreciated.
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Old 02-11-2014, 07:00 AM   #26
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60 pMol is massive! There is obviously something very wrong if you are having to load this much sample to obtain good cluster density. I would recommend starting by quantifying your samples with qPCR to get a better handle on what is actually happening.
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Old 02-11-2014, 07:10 AM   #27
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Quote:
Originally Posted by microgirl123 View Post
60 pMol is massive! There is obviously something very wrong if you are having to load this much sample to obtain good cluster density. I would recommend starting by quantifying your samples with qPCR to get a better handle on what is actually happening.
But do you think that we are underestimating the amounts we are pooling?
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Old 02-11-2014, 07:14 AM   #28
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I'm not sure what I think, but I suspect that either 1. you are not loading as much DNA as you think you are, or 2. your adapters are somehow not right so that your sample is not hybridizing or not priming (you are denaturing before you load onto the MiSeq, right?).

The problem with using the Bioanalyzer to quantify your samples is that it is reading all the DNA present in your sample, whether or not the adapters are attached and whether or not the adapters are correct.
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Old 02-12-2014, 07:18 PM   #29
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I always use the following formula and it never let me down. It considers that the complete length of Your library (on average) is ~350bp (~250bp insert + 100bp adaptors). After pooling of samples (purified using XP beads or alike), I measure concentration using fluorometric assay (qubit or picogreen, high sensitivity).
I adjust the pooled library concentration to 1ng/ul. This gives me a library that is ~4.4nM (considering 350bp average length of pooled library). I take 0.2M of NaOH and mix 1:1 (v/v). This gives me concentration ~2.2nM and 0.1M of NaOH. Next I use hybridization buffer and dilute the library further, 1:150. This gives me a final load of my library ~15pM, NaOH is below 1uM.

NaOH is essential to make the library single stranded (unless You have some adaptors with forks that can be easily separated using 95C incubation/ice and immediate load). The final, low concentration (illumina suggest <1uM) of NaOH is also eseential since it can hinder binding of Your library to a flow cell. Using hybridization buffer to make final dilution is also a must. If You would let us know what is Your protocol right before loading it on machine (with timing for each step) I think somebody might point out some weak spots. I found qPCR methods very disappointing (maybe due to the fact, that I used PhiX dilutions as standards...).
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Old 02-13-2014, 03:12 PM   #30
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Do you already tried the KAPA kit (qPCR) for library quantification? With this kit you quantify only fragments that have the adaptors for the flowcell.
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Old 02-14-2014, 09:28 PM   #31
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Quote:
Originally Posted by liepa View Post
We experience a problem with MiSeq in cluster generation (or cluster detection). We sequence PCR product of a certain gene and the P5 and P7 sequences for the adaptors and dual-indices are added during PCR (pair end).
We pool approx. 350 samples in subpools (with equal conc. of each sample), run on the gel, gel purify, quantify once again and form final pool (with equal conc. of subpools). The final pool quality and quantity looks perfect (for the last run we have purified even twice: 1. gel purification; 2. AMPure PCR purification). The subpools and final pool quantity has been estimated with Agilent 7500 DNA Chip. The first time we loaded on MiSeq 12pMol and the number of clusters formed was 188 with an output of 0.5million reads. We have rerun the same library loading 60pMol, which resulted in 1200 clusters and 5.2G sequences.
Illumina support refuses to help us, as it is self-designed primers.
We have reconsidered all the possible and impossible reasons and looked library preparation and quantification protocols numerous times. So I decided to inquire here, maybe somebody has experienced similar problems or knows what could be the possible explanation for this? Help or insight is really appreciated.
If I read your paragraph properly it looks like you're adding in the P5/P7 sequence into your primer "tail", but that is only the flow cell sequence. What are you using for your sequencing primers? If you have custom primers then that can drastically affect the number of clusters that come up.

FYI -- if you have the updated MiSeq RTA, there is really no reason to use a custom sequencing primer anymore if you load with ~1-5% PhiX. Just add the standard Illumina sequence on top of your PCR primers. This will NOT work with the HiSeq though.
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Old 02-17-2014, 01:38 AM   #32
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Quote:
Originally Posted by bilyl View Post
FYI -- if you have the updated MiSeq RTA, there is really no reason to use a custom sequencing primer anymore if you load with ~1-5% PhiX. Just add the standard Illumina sequence on top of your PCR primers. This will NOT work with the HiSeq though.
I would beg to differ. There are still reasons to run custom primers.
1) You don't need to sequence through the primers, giving you an additional 40-50 bases of sequence per amplicon. This can be vital when designing an optimal primer set.
2) Primers containing P5/7 + index + seq-start-primer + template primer are very long (around 150nt). We have some collaborators that use this approach and they found that not only are the primers expensive to synthesize and produce lower yield, they also seem less robust in terms or reusabliilty - they only survive for a few freeze thaws.

The trick with using custom sequencing primers is to get the Tm up to >65C.
We did this by adding some additional padding sequence between the template primer and our index. This meant we could extend our seq primer on the 5' end for a suitable Tm.
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Old 02-17-2014, 11:38 PM   #33
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Quote:
Originally Posted by TonyBrooks View Post
I would beg to differ. There are still reasons to run custom primers.
1) You don't need to sequence through the primers, giving you an additional 40-50 bases of sequence per amplicon. This can be vital when designing an optimal primer set.
2) Primers containing P5/7 + index + seq-start-primer + template primer are very long (around 150nt). We have some collaborators that use this approach and they found that not only are the primers expensive to synthesize and produce lower yield, they also seem less robust in terms or reusabliilty - they only survive for a few freeze thaws.

The trick with using custom sequencing primers is to get the Tm up to >65C.
We did this by adding some additional padding sequence between the template primer and our index. This meant we could extend our seq primer on the 5' end for a suitable Tm.
I guess my statement was a little too strong. There are a few instances where using a custom sequencing primer would be useful. But it really depends on what you want to accomplish and what kinds of things you value in an experiment.

Keeping the standard sequencing primers means that you don't have to optimize a new sequencing primer. If you're a pro with the Illumina system then that's not really a problem but there are lots of newbies and people who really just don't want to bother with that (especially if you're trying to teach a technician or a graduate student).

Another nice advantage is that you can then multiplex a lot of different types of amplicons together without worrying about differing sequencing primer efficiencies (if you're the kind to blend together two types of sequencing primers anyway). As for the primers themselves, I find it always convenient to do a nested PCR where the inner primers contain the R1/Multiplexing sequencing primer + forward/reverse primers, and use barcoding outer primers (which can be conveniently ordered from NEB). These primers are short and can last a long time.

For some people, the 40-50bp lost using this approach may not be worth it. But sequencing lengths are improving all the time, so I try to not worry about it.
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Old 08-07-2014, 07:45 AM   #34
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Originally Posted by nbogard View Post
Can you please elaborate on this? I am having similar problems with cluster generation on MiSeq but expect that my new libraries with PE adapters will work fine. My concern is whether or not the target for the sequencing primer is also abnormal. I'm worried that two low density runs I have completed (using single-read adapters) consist of useless and misleading data because, in my analysis, I only use the first base immediately downstream from the Read 1 sequencing primer (i.e. where the insert meets the adapter). In my case reads generated beyond that first base would be artifacts.

Do you think the SR adapters only cause a problem with flowcell binding? Would you expect sequencing priming to be normal?
I think I also have similar problem. Can anyone help ?
We recently sent small RNA library prepared using adapters, and primers bought from IDT ( not Illumina kit) for high throughtput sequencing using Miseq. The adapter and primer sequences, and library preparation procedure were adapted from http://www.ncbi.nlm.nih.gov/pubmed/23440203 who sequenced their library on Hiseq. However, we didn't get any reads from our library in Miseq. We verified sequences of our library for the presence of correct adapter and primer sequences via manual cloning/sequencing before sending the library for Miseq. We were in the assumption that what works in Hiseq should work in Miseq too but it seems not after reading the forum. Can anyone help us with the problem ?
We have included a sequence of our library obtained from manual cloning (only top part 5'->3')
AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACGATC------TCACTTCGTATGCCGTCTTCTGCTTG
(------- refers to insert sequence)
We though we included correct P5, P7, and sequencing primers.
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Old 08-07-2014, 08:02 AM   #35
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Quote:
Originally Posted by windhorse8 View Post
I think I also have similar problem. Can anyone help ?
We recently sent small RNA library prepared using adapters, and primers bought from IDT ( not Illumina kit) for high throughtput sequencing using Miseq. The adapter and primer sequences, and library preparation procedure were adapted from http://www.ncbi.nlm.nih.gov/pubmed/23440203 who sequenced their library on Hiseq. However, we didn't get any reads from our library in Miseq. We verified sequences of our library for the presence of correct adapter and primer sequences via manual cloning/sequencing before sending the library for Miseq. We were in the assumption that what works in Hiseq should work in Miseq too but it seems not after reading the forum. Can anyone help us with the problem ?
We have included a sequence of our library obtained from manual cloning (only top part 5'->3')
AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACGATC------TCACTTCGTATGCCGTCTTCTGCTTG
(------- refers to insert sequence)
We though we included correct P5, P7, and sequencing primers.
Your primers are not compatible with the MiSeq. On the HiSeq you can run single read or paired end flow-cells. There are subtle differences between the sequences on the grafted oligos in each flow cell type. The MiSeq uses only paired end sequences on the flow-cell. This means assays designed for single read (HiSeq) flow-cells won't work on the MiSeq. However, paired end libraries will work on both single read and paired end flow-cells.

The single-read and paired end read oligo sequences can be found on page 3 of the supplementary information from the following paper
(http://www.nature.com/nature/journal...ture07517.html)
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Old 08-07-2014, 09:18 AM   #36
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I looked at the sequences for pair-end in the nature paper you referred to. The sequences are different from what we had designed. However, these sequences from the paper are also different from what you would get if you prepare library using Illumina Truseq small RNA sample prep kit ( using the sequence info they provide from their customer sequence letter). When I called Illumina tech support, they said that I can run my small RNA library prepared using their Truseq sRNA kit on Miseq.
I also wonder if Illumina is still keeping the same P5, P7, and sequencing primer sequences as they have shown in the paper back in 2008.
Thank you for your help.

Sidenote: Is there any recent publications who have done small RNA library prep using custom designed primers and adapters and run on Miseq platform? The ones I have come across uses Hiseq.
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Old 08-07-2014, 12:06 PM   #37
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Quote:
We have included a sequence of our library obtained from manual cloning (only top part 5'->3')
AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACGATC------TCACTTCGTATGCCGTCTTCTGCTTG
(------- refers to insert sequence)
There is a note from Illumina that outlines all the sequences that are being used in their library chemistries. Easy enough to find online.

I was going to suggest designing an appropriate custom primer in the black region, but I'm not sure if your P7 region (red) is also too short. But definitely, there is no Miseq compatible sequencing primer in your design.

FWIW.
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Old 08-08-2014, 02:05 AM   #38
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aatgatacggcgaccaccgagauctacac----------------------------tagagcatacggcagaagacgaac
aatgatacggcgaccaccgacaggttcagagttctacagtccgacgatc------tcacttcgtatgccgtcttctgcttg
AATGATACGGCGACCACCGA----------------------------------------AGCATACGGCAGAAGACGAAC
-----------------CGACAGGTTCAGAGTTCTACAGTCCGACGATC


In black, is your library construction.

In red are the sequences on the MiSeq flow-cell (copy & pasted from the Bentley paper). There are a number of differences between those in your assay.
In green are the HiSeq single read flow-cell sequences and those match so your library will cluster on a HiSeq SR flow-cell, but not a HiSeq paired end flow-cell or a MiSeq flow-cell (or a NextSeq flow-cell, for that matter)

In blue is the small RNA R1 sequencing primer which matches. You used to have to add this in as a "custom" primer during sequencing on the GAIIx and HiSeq, I don't know if this is included in the MiSeq primer cocktail, I'm guessing not as the non-TruSeq assay won't cluster on the MiSeq.

The issue is your P5 and P7 sequences. Change these and your assay will work on every Illumina platform.

I recommend looking at the Illumina custom sequence letter (Google it) as it contains all the sequences used in the TruSeq small RNA assay

I should also mention that the MISeq runs hotter than the other Illumina sequencers, so if you are going to use a custom read primer, make sure the Tm >65C. Otherwise, it will anneal badly and cause dim clusters.

Last edited by TonyBrooks; 08-08-2014 at 02:10 AM.
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