SPRI lib prep?

[BIG_SNP]

Does anyone have a protocol for using SPRI beads for the Illumina lib prep? Our lab would like to omit running gels and gel purification steps. I am curious how to utilize the SPRI beads for size selecting fragments instead of running a gel.

Thanks in advance

[Odre]

Hi,

You can find some information on Beckman Genomics website. They launched a new instrument to prepare your library without gel. All is automated (system push and play), even the size fragment selection.

http://www.beckmangenomics.com/produ..._system_i.html

http://www.spriworks.com/

[ECO]

Originally Posted by BIG_SNP

Does anyone have a protocol for using SPRI beads for the Illumina lib prep? Our lab would like to omit running gels and gel purification steps. I am curious how to utilize the SPRI beads for size selecting fragments instead of running a gel.

Thanks in advance

I don't have the reference in front of me, but the Broad just published a paper on automated 454 prep which describes the gel-free size selection protocol. Basically you run an upper and lower size cut with calibrated beads.

Originally Posted by Odre

Hi,

You can find some information on Beckman Genomics website. They launched a new instrument to prepare your library without gel. All is automated (system push and play), even the size fragment selection.

http://www.beckmangenomics.com/produ..._system_i.html

http://www.spriworks.com/

Very cool. Based on the same instrument that Qiagen, Roche, and Invitrogen all have.

Would love to find out more (any?) details of the protocol...something which is clearly missing from the documentation.

[xeno]

Double SPRI for Second Generation Sequencing

Here is a quick protocol:

Please note that depending on your application, it may or may not be necessary to eliminate high MW fragments assuming your shearing profile is relatively tight. And just eliminating the small material will help to increase yields. Make sure when you add the beads you mix well as this has dramatic effects on the yield.

Add 65ul SPRI Ampure beads to 50ul of sample

Mix and incubate for 20 minutes

Separate on magnet (6min), transfer all supernatant (about 115ul) into a new well (off magnet)

Add 100ul of SPRI beads

Mix and incubate for 15 minutes

Separate on magnet (6min), discard supernatant

Wash beads with 60ul of 70% EtOH

Move well off magnet

Dry beads of EtOH (~10min)

Add 40ul EB

Mix and incubate for 3 minutes

Separate on magnet and transfer eluted product into destination plate

[GW_OK]

I've got an exome capture on the sequencer now where I used SPRI beads instead of Qiagen columns. I used 1.8x volume of Ampure XP beads for each cleanup, followed by 500ul 70% EtOH washes and elution in whatever volume of EB I would have eluted for the Qiagen column.
All you're doing is cleaning up enzymatic reactions. Pretend it's just a PCR and clean it up.

[nextgen]

This machine has a few characteritics based on anecdotes I heard so far:
1, you get 10% yield, which means 3 to 4 more cycles of PCR to compensate for the loss. Lib bias may be generated from PCR. 2, The DNA size of the final lib varies from run to run, you will hardly be able to repeat the results. 3, It only does gDNA not cDNA, so that you will still need someone to make lib. By the time they have a cDNA method, you will probably have a different Illumina lib method from what the machine can offer. Overall, if the machine messes up one of our lib, the saving and convenience is gone.
When it is running, it still looks kind interesting with the tips moving up and down. I wish they had a better machine as this particular one is similar to Qiagen's EZ1 biorobot. It is more suitable for larger volume, less precise sample prep, not for this kind of precision oriented task.

Originally Posted by Odre

Hi,

You can find some information on Beckman Genomics website. They launched a new instrument to prepare your library without gel. All is automated (system push and play), even the size fragment selection.

http://www.beckmangenomics.com/produ..._system_i.html

http://www.spriworks.com/

[nextgen]

Use Sizeselector and you will not need double selection. Too much loss of DNA with double selection.

Originally Posted by xeno

Double SPRI for Second Generation Sequencing

Here is a quick protocol:

Please note that depending on your application, it may or may not be necessary to eliminate high MW fragments assuming your shearing profile is relatively tight. And just eliminating the small material will help to increase yields. Make sure when you add the beads you mix well as this has dramatic effects on the yield.

Separate on magnet and transfer eluted product into destination plate

[BasePairMe]

In our lab, the SPRIworks system produces library yields that are very comparable to manual library construction using commercially available reagents. Since only a certain size of fragments are being selected from the sheared DNA, library yields tend to be proportionally lower then what you input to the system. However the automated system recovery is comparable or better then the manual method using a gel cut size selection. There are 3 size options to choose from, one being none. Higher yields result if no size selection is performed during the automated run. We have seen that typically 10 cycles of PCR allows us to enrich enough library for downstream sequencing (Illumina protocols suggest 18 cycles, 10 on some newer protocols).

The size selection on SPRIworks seems very consistent between libraries in the same run and from run to run. (I attached an example of a bioanalyzer trace showing size comparison of 10 libraries) In our hands, it is usually the manual size selection that tends to be somewhat variable based on how the agarose gel runs.

The SPRIworks system is capable of taking any fragmented DNA, whether genomic DNA, cDNA, or PCR products. Since it's a closed automated system, it is not prone to manual errors such as inconsistent pipeting or forgetting to add a reagent.

Based on our initial assessment, the system is fairly precise and consistent with regards to library yield, size distribution, and downstream high quality DNA sequencing – probably the key advantage of the system.

[GW_OK]

What's your cost per library on SPRIworks? More/less/same as Illuminas kits? Can you input 3rd party reagents (ie NEBnext)?

[BasePairMe]

When I consider the time I'm saving since I can do 10 libraries at a time with minimal set up time and the labor costs we're saving I'd have to say it's the same or cheaper then doing them manually. It's so much less work for my team which allows us to concentrate on other things like data analysis. You can't add NEB reagents. The cartridges come fully loaded with library reagents and sealed. The seals are broken by the SPRI-TE instrument in the first step of the automated method. I like this because there is very little chance for user error. The reagents are always the same and we've gotten really consistent results. So far we really like the SPRIworks system. I noticed they are hosting some webinars and you can sign up on the website. www.spriworks.com. The first one is tomorrow.

[nextgen]

It is interesting that I heard of totally different stories.

Originally Posted by BasePairMe

When I consider the time I'm saving since I can do 10 libraries at a time with minimal set up time and the labor costs we're saving I'd have to say it's the same or cheaper then doing them manually. It's so much less work for my team which allows us to concentrate on other things like data analysis. You can't add NEB reagents. The cartridges come fully loaded with library reagents and sealed. The seals are broken by the SPRI-TE instrument in the first step of the automated method. I like this because there is very little chance for user error. The reagents are always the same and we've gotten really consistent results. So far we really like the SPRIworks system. I noticed they are hosting some webinars and you can sign up on the website. www.spriworks.com. The first one is tomorrow.

[gogreen]

I think the cost of the library prep is quite high for the SPRiworks. It would save a lot of hands on time for sure, but I wonder if the throughput that you get for that money is worth it!! The Broad paper says that the amount of the beads that you use for the cleanup is what makes the size selection. Here is the Broad paper

[nextgen]

Cost is an issue. Once you have bought the system, you are lock into the system. You have to keep buying whatever they offer. Another issue is that we all know the the genomics field is evolving surprisingly fast. I don't think their decision making team has the kind of knowledge and experience to keep up the fast speed of genomics field. When new technolgies are coming up, customers have new demand, what do you do? You wait until they finally can offer support after two years? Or you put that little robot into a storage room and make the libraries manually? What if you want to make mutiplex libraries as the Illumina system is becoming ever more powerful?

Originally Posted by gogreen

I think the cost of the library prep is quite high for the SPRiworks. It would save a lot of hands on time for sure, but I wonder if the throughput that you get for that money is worth it!! The Broad paper says that the amount of the beads that you use for the cleanup is what makes the size selection. Here is the Broad paper

[ECO]

Originally Posted by BasePairMe

When I consider the time I'm saving since I can do 10 libraries at a time with minimal set up time and the labor costs we're saving I'd have to say it's the same or cheaper then doing them manually. It's so much less work for my team which allows us to concentrate on other things like data analysis. You can't add NEB reagents. The cartridges come fully loaded with library reagents and sealed. The seals are broken by the SPRI-TE instrument in the first step of the automated method. I like this because there is very little chance for user error. The reagents are always the same and we've gotten really consistent results. So far we really like the SPRIworks system. I noticed they are hosting some webinars and you can sign up on the website. www.spriworks.com. The first one is tomorrow.

These are suspiciously positive reviews for a brand new system, from someone registered with a non-institutional email address.

I'm just saying.

[greigite]

Hi xeno

Do you see any differences in the effectiveness of double SPRI for low vs high concentrations of DNA? These are all volume based measures and I wonder what the effect of concentration is. I have had quite a bit of trouble getting rid of self ligated Illumina adapter at 130 bp using 0.7:1 and 0.75:1 bead:template ratios. Am thinking of testing the effects of template concentration but am curious what the general wisdom is on that.

Originally Posted by xeno

Double SPRI for Second Generation Sequencing

Here is a quick protocol:

Please note that depending on your application, it may or may not be necessary to eliminate high MW fragments assuming your shearing profile is relatively tight. And just eliminating the small material will help to increase yields. Make sure when you add the beads you mix well as this has dramatic effects on the yield.

Add 65ul SPRI Ampure beads to 50ul of sample

Mix and incubate for 20 minutes

Separate on magnet (6min), transfer all supernatant (about 115ul) into a new well (off magnet)

Add 100ul of SPRI beads

Mix and incubate for 15 minutes

Separate on magnet (6min), discard supernatant

Wash beads with 60ul of 70% EtOH

Move well off magnet

Dry beads of EtOH (~10min)

Add 40ul EB

Mix and incubate for 3 minutes

Separate on magnet and transfer eluted product into destination plate

[mccullou]

Originally Posted by ECO

These are suspiciously positive reviews for a brand new system, from someone registered with a non-institutional email address.

I'm just saying.

ECO- I was thinking the same thing

For those that have the SPRI, perhaps I am missing something in the literature, but does this instrumentation do the complete library prep from properly sized DNA (clean/As/Lig/Amp) and produces libraries ready for clustering, aside from QC of course?

Additionally, is there a module for multiplexing, and if not, how would it work with existing reagents/programs?

[nilshomer]

Originally Posted by BasePairMe

In our lab, the SPRIworks system produces library yields that are very comparable to manual library construction using commercially available reagents. Since only a certain size of fragments are being selected from the sheared DNA, library yields tend to be proportionally lower then what you input to the system. However the automated system recovery is comparable or better then the manual method using a gel cut size selection. There are 3 size options to choose from, one being none. Higher yields result if no size selection is performed during the automated run. We have seen that typically 10 cycles of PCR allows us to enrich enough library for downstream sequencing (Illumina protocols suggest 18 cycles, 10 on some newer protocols).

The size selection on SPRIworks seems very consistent between libraries in the same run and from run to run. (I attached an example of a bioanalyzer trace showing size comparison of 10 libraries) In our hands, it is usually the manual size selection that tends to be somewhat variable based on how the agarose gel runs.

The SPRIworks system is capable of taking any fragmented DNA, whether genomic DNA, cDNA, or PCR products. Since it's a closed automated system, it is not prone to manual errors such as inconsistent pipeting or forgetting to add a reagent.

Based on our initial assessment, the system is fairly precise and consistent with regards to library yield, size distribution, and downstream high quality DNA sequencing – probably the key advantage of the system.

As another user reported, is "your lab" in fact "Beckman Coulter":
http://www.beckmangenomics.com/docum..._SPRIworks.pdf.
(Your figure above is on the 2nd page).

[ECO]

Originally Posted by nilshomer

As another user reported, is "your lab" in fact "Beckman Coulter":
http://www.beckmangenomics.com/docum..._SPRIworks.pdf.
(Your figure above is on the 2nd page).

+1 for ECO.

[zacz]

Performing a standard Ampure cleanup with 0.8X beads will remove adaptor dimers and small fragments; double sided is usually overkill in my opinion because you will never get tight enough for paired end sequencing and large fragments will only give you a little heterogeneity in cluster size (which really shouldn't hurt much). As someone else mentioned, it's easier to fine tune your range during shearing

[RUmustang]

Originally Posted by xeno

Double SPRI for Second Generation Sequencing

Here is a quick protocol:

Please note that depending on your application, it may or may not be necessary to eliminate high MW fragments assuming your shearing profile is relatively tight. And just eliminating the small material will help to increase yields. Make sure when you add the beads you mix well as this has dramatic effects on the yield.

Add 65ul SPRI Ampure beads to 50ul of sample

Mix and incubate for 20 minutes

Separate on magnet (6min), transfer all supernatant (about 115ul) into a new well (off magnet)

Add 100ul of SPRI beads

Mix and incubate for 15 minutes

Separate on magnet (6min), discard supernatant

Wash beads with 60ul of 70% EtOH

Move well off magnet

Dry beads of EtOH (~10min)

Add 40ul EB

Mix and incubate for 3 minutes

Separate on magnet and transfer eluted product into destination plate

Xeno, thanks for your protocol. I am wondering if you could tell how much initial DNA (in 50uL) you usually put in in the first round selection? what's the size of the fragment you get after the second round selection? could you please post bioanalyzer profile?