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  • #16
    We are buying reagents for smart-seq2. It turns out that TSO is not very cheap(~750usd for the smallest scale 250nmole)? Meanwhile I want to make sure there is nothing wrong with my technique otherwise will face the same problem in the future. Any idea what could possibly go wrong?

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    • #17
      strange cDNA profile after SMART-Seq2

      Hi, Simone, we are testing SMART-seq2 protocol in our lab recently. It's quite impressive in terms of cDNA yield after preamplification. However, we notice that the our cDNA library is not as uniform as it should be. Although we can see it covers the range from several hundred to thousands, there are several intense peaks with the highest of around 1800bp.
      We tried different TSO concentration and cycle numbers but did not see any improvement. Please see attachment 1 (cDNA is purified prior to bioanalyzer analysis)
      We also tried to replace some component or add additional bead purification after first strand synthesis. The oligo dT30 primer provided in the clontech smart-seq kit did show some improvement while there are more concatmer formation with additional bead purification step. Please see testing result 2 (We want to see whether there is concatemer formation so we load the PCR product in bioanalyzer without purification).

      I think I'm close to a successful run but also so desperate that I can not solve this problem. Could anyone please give me some suggestions? What are these bands and how to get rid of them? Thanks a lot.

      Originally posted by Simone78 View Post
      we have recently published a paper where we describe Smart-seq2, an improvement of the Smarter kit. Check it out! You don´need any kit to make your cDNA and you´re going to save a lot of money!

      Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multi …

      http://www.ncbi.nlm.nih.gov/pubmed/24056875
      Attached Files

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      • #18
        Hi,
        what you saw here is nothing new for us and we still can´t explain what it is. we also saw a peak around 1.8 Kb when optimizing the protocol using HEK293T (human) cells. When we looked for over-represented sequences in the data we found out that the peak corresponded to a specific transcript called "humanin", which is mitochondrial (!) but has several homologous copies on the genome. While it´s annoying having such a library, the results were not affected. Below please find an example of what we got when we were optimizing Smart-seq2. There you can see we had not only this humanin peak but also a fair amount of primer dimers, but still the performance of our method was superior to the Clontech kit (primer dimers get tagmented with the Nextera kit as well and end up in the final library, thus "wasting" sequencing reads). Sometimes we see it also with mouse cells, but this obviously can´t be humanin which, as the name says, is present only in humans. For the mouse we never found out what it is...what we know is that it´s not a contamination because some of our collaborators reported the same. On the other hand, even when working with single cells this peaks doesn´t always come up. Maybe it depends on how stressed the cells are or how they were sorted? I would also be interested in finding it out!
        In conclusion, Smart-seq2 is better than Smart-seq but it´s not perfect, sorry!

        I saw that you tried to reduce the amount of TSO. I did few trials on the amount of the different primers. While reducing ISPCR and SMART dT30VN sometimes (sometimes!) helps in reducing the amount of primer dimers, decreasing the TSO usually leads to lower cDNA yield after preampl. Maybe the strand-switch reaction is inefficient and you need to have such a huge amount of TSO even when working with single cells!
        Have you tried to sequence some of these libraries? I don´t know what you are interested in, but if you need a lot of reads you might simply multiplex less samples per lane. For most applications (diff expression, detection of isoforms/splice variants) 1 million reads/sample are sufficient and you would get that amount even when pooling 96 samples on a Illumina HiSeq 2000.

        We had some concatamer problem in the beginning, but they were due to too much adaptors (TSO, ISCR or oligo dT) compared to the RNA of a cell and we observed that only with very small cells (mostly immune cells which have very little mRNA). If it is an issue, you might try to block the TSO at the 5´end as done by Kapteyn J et al (BMC Genomics 2010, 11:413). Blocking the 5´should prevent concatamerization of the TSO.

        Best,
        Simone
        Last edited by Simone78; 08-24-2017, 05:42 AM.

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        • #19
          Maybe one possible problem is the LNAs in the TSO? This is prone to strand invasion (doi:10.1186/1471-2164-14-665), so the LNAs are not a good idea if you care about full-length transcripts. That's why all the other template-switching protocols use riboguanines. If the strand invasion is biased toward specific sites, that could explain these problems.

          EDIT: on further inspection, those bands are definitely the characteristic "hedgehog" signal from using unblocked oligos. Block them and it should be fine. I don't know why there are any protocols floating around out there that say blocking is optional; it's so cheap to fix.
          Last edited by jwfoley; 06-02-2014, 06:35 AM.

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          • #20
            Originally posted by jwfoley View Post
            Maybe one possible problem is the LNAs in the TSO? This is prone to strand invasion (doi:10.1186/1471-2164-14-665), so the LNAs are not a good idea if you care about full-length transcripts. That's why all the other template-switching protocols use riboguanines. If the strand invasion is biased toward specific sites, that could explain these problems.

            EDIT: on further inspection, those bands are definitely the characteristic "hedgehog" signal from using unblocked oligos. Block them and it should be fine. I don't know why there are any protocols floating around out there that say blocking is optional; it's so cheap to fix.
            We use a LNA-containing TSO and always have a better coverage at the 5´-end of transcripts than the Clontech kit which uses 3 riboG.

            In our hands, blocking the TSO led to a (slightly) lower gene detection at all RPKM levels and a (slightly) higher variation between replicates. The unblocked LNA-containing TSO was still performing better, therefore we don´t block the TSO unless the "hedgehog" signal shows up.

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            • #21
              We use a LNA-containing TSO and always have a better coverage at the 5´-end of transcripts than the Clontech kit which uses 3 riboG.
              Not a fair comparison since your protocol has several other optimizations relative to the Clontech kit (or just its legacy version, I assume).

              The unblocked LNA-containing TSO was still performing better, therefore we don´t block the TSO unless the "hedgehog" signal shows up.
              Is it realistic to buy two versions of the oligo, and risk losing precious samples, just to squeeze out a slight performance gain? I would rather stick to the most robust version of the protocol.

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              • #22
                Originally posted by jwfoley View Post
                Not a fair comparison since your protocol has several other optimizations relative to the Clontech kit (or just its legacy version, I assume).
                You´re right. But if you check in the Suppl Info of our Nat Methods paper you will see different comparisons. We singled out the effect of the polymerase and the TSO and showed, among other things, that LNA-based TSO is better than rG3-based TSO, all the other factors IN THE SMART-SEQ2 PROTOCOL kept the same.

                Originally posted by jwfoley View Post
                Is it realistic to buy two versions of the oligo, and risk losing precious samples, just to squeeze out a slight performance gain? I would rather stick to the most robust version of the protocol.
                These days we FACS-sort most of our cells. Every cell type requires few adjustment for optimal results (amount of PCR primers, number of cycles, etc). We always have to test few of them anyway once we start a new project. Only if the TSO creates concatamers we switch to the iso-TSO. The most robust version of the protocol is still the one with the unmodified version of the LNA-TSO (at least for us).

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                • #23
                  We singled out the effect of the polymerase and the TSO and showed, among other things, that LNA-based TSO is better than rG3-based TSO, all the other factors IN THE SMART-SEQ2 PROTOCOL kept the same.
                  If I'm reading this right (doi:10.1038/nmeth.2639, supplementary table 1, sheet B, rows 34 and 35) your LNA TSO yielded 7% shorter cDNAs than your pure RNA TSO, and 11% shorter than the SMARTer TSO, on average. This is consistent with my suspicion that the gain in yield is due in part to the TSO's mispriming in the middle of the cDNA by strand invasion.

                  These days we FACS-sort most of our cells. Every cell type requires few adjustment for optimal results (amount of PCR primers, number of cycles, etc). We always have to test few of them anyway once we start a new project. Only if the TSO creates concatamers we switch to the iso-TSO. The most robust version of the protocol is still the one with the unmodified version of the LNA-TSO (at least for us).
                  I guess we have different ideas of what robustness should be. To me, it means any inexperienced lab can perform the protocol as written and see it work well the first time. Reoptimizing every time, fallback versions, etc. are okay for large service centers or labs that do the same protocol a lot, but they discourage wide adoption of the method.

                  Also, you mention using iso nucleotides as your 5' blocker. Have you done the test using biotin instead? That seems more common in the literature, and is a lot cheaper (plus it's difficult to get LNAs and iso bases from the same company). In my experience it works just fine to eliminate the "hedgehog".

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                  • #24
                    Originally posted by jwfoley View Post
                    If I'm reading this right (doi:10.1038/nmeth.2639, supplementary table 1, sheet B, rows 34 and 35) your LNA TSO yielded 7% shorter cDNAs than your pure RNA TSO, and 11% shorter than the SMARTer TSO, on average. This is consistent with my suspicion that the gain in yield is due in part to the TSO's mispriming in the middle of the cDNA by strand invasion.
                    A possibility that can´t be excluded and a phenomenon that is happening for sure to some extent. I already said the protocol is not perfect...
                    What we wanted to show with that table as well as Suppl Figure 1, 3 and 5 (among others) is the synergic effect of betaine, MgCl2 and LNA-TSO. LNA-TSO was chosen because it improved the efficiency of the strand switch reaction, a limintations when using rG3. Undesired phenomenons are possible, though.

                    Originally posted by jwfoley View Post
                    I guess we have different ideas of what robustness should be. To me, it means any inexperienced lab can perform the protocol as written and see it work well the first time. Reoptimizing every time, fallback versions, etc. are okay for large service centers or labs that do the same protocol a lot, but they discourage wide adoption of the method.
                    Yes, I guess we have different ideas. If you would have tried the protocol yourself instead of just looking for mistakes or inaccuracies you would have realized how robust the protocol is. I didn´t talk about a complete makeover of the protocol each time you start with a new cell type. I talked about few adjustments which won´t take more than half a day. On the other hand, cells are different in terms of RNA content (to mention one factor) which requires a different number of cycles and amount of primers in order to avoid over/under-amplification, excess of primer dimers etc. I guess that people out there are able to do this without following a protocol (but we also mention it, if you would have read the Nat Protocols paper you would have noticed (step 14). To my knowledge, many groups have already switched to Smart-seq2, got good results and are happy with that. Verba volant scripta manent , to say it in Latin.

                    Originally posted by jwfoley View Post
                    EDIT: on further inspection, those bands are definitely the characteristic "hedgehog" signal from using unblocked oligos. Block them and it should be fine. I don't know why there are any protocols floating around out there that say blocking is optional; it's so cheap to fix.
                    Originally posted by jwfoley View Post
                    Also, you mention using iso nucleotides as your 5' blocker. Have you done the test using biotin instead? That seems more common in the literature, and is a lot cheaper (plus it's difficult to get LNAs and iso bases from the same company). In my experience it works just fine to eliminate the "hedgehog".
                    Originally posted by jwfoley View Post
                    Is it realistic to buy two versions of the oligo, and risk losing precious samples, just to squeeze out a slight performance gain? I would rather stick to the most robust version of the protocol.
                    Not really clear to me what you suggest...Do you want to block it or not? The "most robust version" according to what you say is the 3rG which is not blocked...but then you suggest biotin as blocking group...I´m a bit confused! Besides, biotin as well as 2-OMe group, phosphates, etc don´t make things better, at least in our hand...I would be really interested in seeing biotin working as a blocking group in this context, if you have results showing that...
                    Best,
                    Simone

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                    • #25
                      If you would have tried the protocol yourself instead of just looking for mistakes or inaccuracies you would have realized how robust the protocol is.
                      I did try it, and I spent a couple of weeks testing various things before I ran it on the Bioanalyzer. Then I saw the hedgehog pattern, and realized I had wasted all that time optimizing my yield of concatamers, so I ordered biotin-blocked oligos, waited another week for them to arrive, and moved on.

                      Not really clear to me what you suggest...Do you want to block it or not? The "most robust version" according to what you say is the 3rG which is not blocked...but then you suggest biotin as blocking group...I´m a bit confused! Besides, biotin as well as 2-OMe group, phosphates, etc don´t make things better, at least in our hand...I would be really interested in seeing biotin working as a blocking group in this context, if you have results showing that...
                      Sorry if that was confusing. Yes, I always use a blocking group now, but I use biotin instead of unnatural bases because it is much cheaper. I was getting the "hedgehog" fairly consistently without the biotin, and I have never seen it with the biotin, though so far I've only run a few dozen samples of either type on the Bioanalyzer.

                      Here is the test that convinced me not to do any more preps with unblocked oligos:

                      Unfortunately we had a bad lot of Bioanalyzer reagents (this seems to be a common problem), so the markers ran too slowly in the first wells, but you can clearly see a hedgehog pattern in every sample where I used unblocked primers. Post-PCR, those peaks totally overload the Bioanalyzer. Even the water (no template) control doesn't have a hedgehog when I use biotinylated oligos - at least not before PCR.
                      Last edited by jwfoley; 06-08-2014, 05:26 AM.

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                      • #26
                        I think we are talking about 2 different things.
                        What you have in your Bioanalyzer when you use unblocked oligos looks to me like primer dimers (and not longer concatamers) which are a problem anyway and it´s interesting to see it works for you. We also have such a problem, but usually the final results are not affected. Yes, this "short stuff" gets tagmented by the tn5 and we waste reads sequencing it but it´s never too bad.
                        Since we also make our own Ampure beads I just changed the % of PEG in the bead buffer, because the % of PEG directly correlates with the beads cutoff. In this way we reduce even more the leftover dimers in the final library.
                        Anyway, what I mean when I talk about "concatamers" is a profile that looks more like the one I show here. Sample 5 and 6 have a "moderate" amount of concatamers, while samples 9 and 10 are much worse.

                        Btw, your post-PCR with biotinylated samples aren´t looking too good. You have a lot of short stuff between 60 and 90 sec that shouldn´t be there (premature termination of RT? degradation?). Have you sequenced any single cell using this protocol? I guess these libraries have a strong 3´-bias.
                        Last edited by Simone78; 08-24-2017, 05:42 AM.

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                        • #27
                          What you have in your Bioanalyzer when you use unblocked oligos looks to me like primer dimers (and not longer concatamers)
                          The smallest peak, which I also get from the blocked oligos, is primer dimer. Larger peaks must be concatamers (primer multimers), since I see them even when I do the TS-RT with no RNA template. You can call them whatever you want, but they contain no useful cDNA sequence and they get above the size range that I can safely size-select out (and at very high concentrations, apparently), so I strongly prefer the blocked oligos to keep it clean. Maybe you get the wider range of concatamer sizes because you're using the LNA TSO?

                          You have a lot of short stuff between 60 and 90 sec that shouldn´t be there (premature termination of RT? degradation?). Have you sequenced any single cell using this protocol? I guess these libraries have a strong 3´-bias.
                          These particular samples contained fragmented RNA, so that's expected.

                          No, I haven't brought it down to single-cell amounts of input yet. But if I do, I may not have the choice to test one batch of expensive oligos to see if it works, and then switch to a different batch of expensive oligos if it doesn't, on the same sample. So I'm only using blocked oligos for every sample.
                          Last edited by jwfoley; 06-08-2014, 02:45 PM.

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                          • #28
                            Originally posted by jwfoley View Post
                            No, I haven't brought it down to single-cell amounts of input yet. But if I do, I may not have the choice to test one batch of expensive oligos to see if it works, and then switch to a different batch of expensive oligos if it doesn't, on the same sample. So I'm only using blocked oligos for every sample.
                            the cost/sample of the "batch of expensive oligos" is negligible...you don´t have to order buckets of it...
                            it would be interesting to see the gene body coverage of your TSO compares with the kit and with a "batch of expensive oligo".
                            Since we made the protocol so cheap I think I´ll stick to a batch of expensive oligos for now, at least until you prove the rest of the world wrong...good luck with the tests!

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                            • #29
                              the cost/sample of the "batch of expensive oligos" is negligible...you don´t have to order buckets of it...
                              Oh, maybe that's why we see this differently. With my supplier, the smallest possible order of the TSO is about 50 micrograms, for about USD 500. Obviously that's enough for hundreds or thousands of libraries, but if you only want to make a dozen, it's a pretty big commitment, especially when the rest of the reagents are off-the-shelf and/or can be bought in much smaller effective amounts. So doubling the amount of TSO you must purchase, just to try a risky way of getting a slight yield increase that may require falling back to the foolproof way anyhow, is rather unattractive if you only plan to do one experiment rather than start a service center. Otherwise you might just buy the kit.

                              at least until you prove the rest of the world wrong
                              I don't know what the rest of the world does, but the STRT-seq folks aren't even outside your institution: "Critical: This oligo must be 5′ biotinylated to prevent template switching at its 5′ end (which would generate useless background reads)." (doi:10.1038/nprot.2012.022) At any rate, your own protocol's note that unblocked oligos sometimes result in concatamers is more than enough to persuade me not to waste any time on them.

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                              • #30
                                Originally posted by jwfoley View Post
                                I don't know what the rest of the world does, but the STRT-seq folks aren't even outside your institution: "Critical: This oligo must be 5′ biotinylated to prevent template switching at its 5′ end (which would generate useless background reads)." (doi:10.1038/nprot.2012.022) At any rate, your own protocol's note that unblocked oligos sometimes result in concatamers is more than enough to persuade me not to waste any time on them.
                                I suggest you reading both papers from the Linnarsson group once again (the Genome Research 2011 and the Nature Protocols 2012). Neither of them has a biotinylated TSO, don´t know where you read it...
                                In the Nat Prot paper the biotinylation refers to the oligodT primer (STRT-V3-T30VN oligo), while in the Genome Research paper they biotinylated both the STRT-V3-T30VN and the STRT-PCR primer (to capture cDNA later on). It obviously works for them but it was less successful for us (because of the different TSO? I don´t know). That´s why I was trying to block the TSO instead.

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