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  • #16
    Originally posted by ECO View Post
    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?
    Last edited by mccullou; 04-09-2010, 02:11 PM. Reason: typo

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    • #17
      Originally posted by BasePairMe View Post
      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).

      Comment


      • #18
        Originally posted by nilshomer View Post
        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.

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        • #19
          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

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          • #20
            Originally posted by xeno View Post
            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?

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            • #21
              SPRIworks from Beckman Coulter

              Let me comment on a couple of posts below with regards to the SPRIworks system from Beckman Coulter. Firstly, Beckman Coulter wouldn't have placed 25+ SPRIworks systems across the world with well known genomics centers if the system would not be reliable and reproducible as mentioned by others here. It is actually the opposite using SPRIworks results in at least similar to manual but in most cases better sequencing data quality.
              Especially if it comes to Illumina the system is very versatile and can be used for a large variety of applications like RainDance selected libraries, std single and PE libraries, SureSelect enriched libraries downstream, cDNA libraries, second part of RNA-Seq libraries, etc. The system is also flexible with regards to the amount of starting material between 5µg and 10ng which again makes it very versatile. So, this is no theory this is reality.
              On top of that libraries for the GS FLX can be produced as well and others will follow over the course of the next weeks.

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              • #22
                I wonder who is working on automated library construction that is an open system so that you don't have to be tied up to a particular vendor. Some institutes have their own high throughput library construction system. However, they don't share. If anyone is working on automated library construction, I am interested in licensing it. I am thinking about making a open source automated library construction system that can be ported to different robotic systems and people can use whatever suitable reagents they like. Ideally, it will make 1 to 8 up to 32 libraries per run. 96-library prep system probably requires fairly large reservoirs. Please PM me if you are willing to share, we can discuss more in detail. The goal is to come up with a term that will benefit the original creators as well as the research community as a whole. Any idea is welcome.
                Last edited by nextgen; 09-06-2010, 09:25 AM.

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                • #23
                  Here:
                  http://www.genome.ou.edu/proto.html

                  Every library prep system is "open" now. All you need is the individual reagents (which you can get from multiple sources) and the oligos (which you can also get from multiple sources). Look at NEB and IDT for instance. All the robot does is pipette the reagents and do an SPRI cleanup after the reaction. And automating an SPRI cleanup isn't that hard.

                  All you need is a robot and someone with the knowledge and time to set it up. I don't know why you'd need to license it.

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                  • #24
                    It is true that all the robots do is pipetting. However, if someone has spent the time writing up scripts and user interface and tuning up the process, it will really save a lot time and effort to make adaptation. The creator of the cripts should get compensated for that, I think, especially when people are reluctant want to share for free. I am willing to chip in to accelerate the process. I have talked to a few people. They are very positive. It should be very easy to port script. It can be fully automated and adjusted based on daily need. It can be very user friendly. It is in the work.
                    Last edited by nextgen; 09-06-2010, 09:03 AM.

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                    • #25
                      The SPRI-Works is a good system for what it does. I have had one in the lab for several months and have seen very consistent performance and reproducibility. The yield is a little low but the size selection is one of the best implementation of beads that I have seen. The 200-400 selection is very effective. The 300-600 leaves a little to be desired. The inconsistencies that may be observed are likely to do more with inconsistent sonnication rather than instrument. The instrument performs all reaction steps at RT and actually has a very elegant way of handling the reagent tubes for both reactions and purifications. Overall, I think Beckman has done a great job.

                      That said, the instrument isn't perfect but it is robust. The reagents are slightly on the pricey side, especially since adaptors are not included. The ability to run 1-10 libraries at the same time is a good feature for low throughput.

                      There are also a number of situations where it is not an ideal system such as preparing libraries for sequence capture. cDNA or gDNA are both fine since you can always select 'no size selection' if needed.

                      One thing that is interesting is how it does the size selection. It is a very elegant solution that isn't like the other protocols that are out there using beads now (double-selection, etc).

                      We also perform library preps using two Beckman FX-P robots. One of the FX-Ps has an on-deck thermocycler along with shaking and static peltiers. The other FX-P has a tip wash station. We have fully automated the library prep process on the thermocycler-equipped version, including size selection (assuming covaris-sheared DNA). It works extremely well and we routinely do 96-well library prep for high-throughput exome capture projects. That said, it is faster to perform the library prep using 12-channel pipettes and use the robot to do the purification steps but the hands on time is obviously longer.

                      The challenge with sharing scripts is that unless your FX-P (or other robot of choice) is equipped exactly the same with the same deck layout and you use the same consumables, it would be impossible to use other's scripts. Anyone with the skills and comfort to adapt a developed script to their deck would probably write their own anyway.
                      HudsonAlpha Institute for Biotechnology
                      http://www.hudsonalpha.org/gsl

                      Comment


                      • #26
                        Library construction on an open LiHa system

                        An open system can certainly be an alternative but there is a couple of things to be considered. Firstly a question: can an open system really cover all the steps in order to fully automate the entire process? Secondly, if you have a standard pipeline (always the same application, high numbers of samples) it may be worth looking into it but not if your applications vary from day to day. Thirdly, implementing protocols can take you weeks/months in the worst case. Fourthly, most likely there is high investment cost associated with such a system, especially if you want it to cover the entire process. Lastly, it has been reported that such systems have a high tendency towards cross contamination.
                        Taken all this together, you need to think twice which road you want to take, right? Ultimately, the quality of the library impacts the quality of the sequencing data.

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                        • #27
                          I'm hoping to go gel free for all of my library preps (I've had some cross contamination issues that I think is coming from the gel step) and would love to try the double spri method.

                          I've used SPRI beads only for my RNAseq preps and it works great but haven't had the same success for my ChIPseq preps, which is likely due to the bigger size distribution of fragments from shearing.

                          Has anyone had any success with the double SPRI method or does anyone know a good protocol?

                          Thanks!

                          Chris.

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                          • #28
                            hey,

                            I do double size selection using SPRI. The results are not consistent.
                            I sometimes get bigger fragments. I wounder if I am carrying some beads in the first selection. Does anyone use 96 well manually. My yield is around 30%.
                            This how I do.
                            i fragment DNA using covaris.
                            Then separate bigger fragments and then smaller fragments selcting the size I want.
                            Does any one use this way and have similar yields?

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                            • #29
                              SPRI for DNA< 70bp

                              Hi everyone,

                              I am trying to make a library from DNA degraded to <70bp. Would the "no size selection" SPRI bead perform better than the MinElute kit? I don't have HMW DNA, only very small.
                              I'm at loss here. It would be a pitty to lose lots of DNA during the purifications that come after the blunt end and adenylation steps of the library prep.

                              Comment


                              • #30
                                Originally posted by odile View Post
                                Hi everyone,

                                I am trying to make a library from DNA degraded to <70bp. Would the "no size selection" SPRI bead perform better than the MinElute kit? I don't have HMW DNA, only very small.
                                I'm at loss here. It would be a pitty to lose lots of DNA during the purifications that come after the blunt end and adenylation steps of the library prep.
                                I don't think you can use the standard Ampure cleaning protocol, it removed most of the <75 bp fragments when I used it. 70bp DNA in combination with ~75bp adapters might work however, see attachment.
                                Attached Files
                                Last edited by JUdw; 03-23-2011, 01:14 AM.

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