Tag: RNAi screening

RNAi screening: a powerful tool for gene function discovery and therapeutic target identification.

Similar seed effects in independent siRNA screens

Similar seed effects in independent siRNA screens

A 2013 study on Parkin translocation used genome-wide siRNA libraries from Ambion (single Silencer Select siRNAs) and Dharmacon (pools of 4 siGENOME siRNAs).

The correlation between results for the same on-target gene from the two libraries was very low (R = 0.09). (Each point in the following plot is for a gene.)

% Parkin Translocation (PPT) for Ambion vs. Dharmacon siRNAs grouped by same 7mer seed

The correlation between results for the same 7mer seed were higher (0.26), providing another example of the Iron Law of RNAi Screening. (Each point in the following plot is for a 7mer seed.)

It is also worth noting that the seed-based correlation would likely have been much higher, had the Dharmacon siRNAs been screened individually (see details below).

Conclusion

The only effective way to avoid off-target effects in RNAi screening is to use high-complexity reagents like siPOOLs, which dilute away off-target effects while maintaining strong on-target silencing.

Analysis details

To calculate the Ambion by-gene value, the mean PPT value was taken for the 3 on-target siRNAs for the gene. (The Dharmacon pooled library only has 1 value per gene, so no further calculation is necessary.)

To calculate the Ambion by-seed value, the mean PPT value was taken for all siRNAs with the 7mer. For Dharmacon, the pool value was assigned to each siRNA, and then siRNAs were grouped by their 7mer seed in order to calculate the seed mean. This means that the Dharmacon siRNA seed value is actually the average from 4 different siRNAs (with different seeds). Had the Dharmacon siRNAs been screened individually, the correlation with Ambion seed results would have been higher.

The Iron Law of RNAi Screening

The Iron Law of RNAi Screening

This is the lead singer of a band called Iron Law. He looks like a researcher experiencing massive frustration after discovering what we call the Iron Law of RNAi Screening.

This law states that in any screen with low-complexity reagents (single siRNAs like Silencer Selects, or mini-pools like Dharmacon SMARTpools), off-target effects will predominate.

Given that the average lone siRNA will down-regulate nearly 100 off-target genes, but has only a single on-target gene, it is not hard to see how this comes about.

The only effective way to break this law is to use high-complexity reagents like siPOOLs, which dilute away off-target effects while maintaining strong on-target silencing.

Below is a figure showing the reduced off-target effects with a siPOOL (3 nM) after 48 hours in HeLa cells:

Reduce Off Targets effect with siPOOLs

Transcriptome-wide profiling revealed a single siRNA can induce numerous off-targets (red dots) while a  siPOOL against the same target gene (green dot), and containing the non-specific siRNA, had greatly reduced off-target effects.

siTOOLs Biotech sponsors Argonautes Conference 2022

siTOOLs Biotech sponsors Argonautes Conference 2022

MUNICH, GERMANY – siTOOLs Biotech GmbH, a young, research-driven biotech company based in Munich/Martinsried and rapidly growing in the field of functional genomics and Next-Generation RNA sequencing, today announced its support for the upcoming Argonautes Conference 2022 at University of Regensburg as lead sponsor.

The conference, organized by Prof. Gunter Meister from University of Regensburg, and Prof. Martin Simard of CHU de Québec-Université Laval Research Center, will be held from 24th to 27th August 2022.

The conference’s scientific program will focus on Argonaute, the core protein of the RNAi mechanism: and how mutations in the Argonaute gene can lead to rare genetic disorders.  The program starts with sessions dedicated to the diverse roles of prokaryotic Argonaute proteins (pAgo), and the functions of Argonaute in plants. It will then move to new insights in the structure of Argonaute proteins, touch on the role of Argonaute in the silencing of transposable elements and show how Argonaute is involved in animal development. The conference will finally come to the 4 human Argonaute proteins and their role in disease.

Andrew Walsh, bioinformatician at siTOOLs Biotech is one of the confirmed speakers of the event. He will show how large-scale RNAi screening datasets can be used to study the molecular behavior of human Argonaute proteins. Other highlights include a keynote lecture by Dr. Leslie Gordon, who drove the development of a treatment for a rare disease Progeria, and a panel with affected families sharing their experiences.

“We are excited to sponsor the Argonautes Conference,” said Michael Hannus, Founder and Managing Director of siTOOLs Biotech. “As an RNA company, our mission has always been to support and facilitate RNA research to help accelerate scientific breakthroughs. We are sponsoring this conference to promote the research and drive awareness of Argonaute related diseases. Hopefully the conference can contribute to finding solutions for the life of children and families impacted by AGO2 (Lessel-Kreienkamp/Leskres) syndrome.”

Are you doing RNAi research, arrayed RNAi screens, or CRISP validations?

Come by and ask us any questions regarding your experimental setup, methods or bioinformatics analysis.

Connect with us through this link to receive updates and meeting opportunities for siTOOLs Biotech at the conference. We will be setting up a booth at this event where you can meet our team of scientists!

References:

Featured image: Regensburg, from mojolo/Adobe Stock

Low hit validation rate for Dharmacon siGENOME screens

Low hit validation rate for Dharmacon siGENOME screens

Good experimental design is important when validating hits from RNAi screens.  Off-target effects from single siRNAs and low-complexity siRNA pools (e.g. Dharmacon siGENOME) result in high false-positive rates that must be sorted out in validation experiments.

Dharmacon siGENOME pools (SMARTpools) have 4 siRNAs, and the most common form of validation is to test the pool siRNAs individually (deconvolution).

Unfortunately, the results of such deconvolution screening rounds are difficult to interpret.

The pool phenotype could be due to the off-target effects of any single siRNA, or even synthetic off-target effects from pooled siRNAs.

Rather than deconvoluting the pool, a better approach is to test with independent reagents.  Should the phenotype be due to the seed effects of an siRNA in the siGENOME pool, the new designs (with presumably different seed sequences) should not show them.  (Note that because they have their own potential complicating off-targets, an even better option would be to use a reagent like siPOOLs that minimises the likelihood of off-target effects).

Independent validation reagents was the approach used by Li et al. in a screen looking for enhancers of antiviral protein ZAP activity.

They first did a genome-wide (18,200 genes) screen with siGENOME pools, looking for pools that increased viral infection rate.

The biggest effect was with the positive control, ZAP (aka ZC3HAV1).   Several other pools also stood out as giving large increases in viral infection (Fig 1B):

They identified 90 non-control genes with reproducible Z-scores above 3 in their replicate experiments (~0.5% of screened genes).

These 90 genes were then tested with 3 Ambion Silencer siRNAs.  (They also included a few genes in the validation round based on pathway information and off-target analysis– more on this below.)

Of the 90 candidate hit genes, only 11 could be confirmed (Fig 2B, note that ZC3HAV1/ZAP is the positive control and JAK1 was added to the validation round based on pathway info.  A gene was considered confirmed if 2 of 3 siRNAs had a Z-score > 3.):

 

We also see that only 1 of the 7 top hits from the first round (blue genes in the first figure) was confirmed.  This is a common observation in RNAi screens: the strongest phenotypes are mostly due to off-target effects.

Off-target effects are difficult to interpret, even using advanced analysis programs like Haystack or GESS.  The authors tested 4 genes identified by Haystack as targets for seed-based off-targeting.  None of those genes could be confirmed in the validation round.

Pooling only 4 siRNAs increases off-target effects

Pooling only 4 siRNAs increases off-target effects

In a previous post, we showed how siRNA pools with small numbers of siRNAs can exacerbate off-target effects.

Low-complexity pools (with 4 siRNAs per gene) should thus lead to overall stronger off-target effects than single siRNAs.

This phenomenon was addressed in a bioinformatics paper a few years back.  The authors created a model to predict gene phenotypes based on the combined on-target and off-target effects of siRNAs.

The siRNAs were screened either individually (Ambion and Qiagen), or in pools of four (Dharmacon siGENOME), in 3 different batcterial-infection assays (B. abortus, B. henselae, and S. typhimurium).

The model assumed that each siRNA silenced its on-target gene to the same level.  For off-target silencing, they used the predictions from TargetScan, a program for calculating seed-based knockdown by miRNAs or siRNAs.

In order to assess model quality, they checked how similar the gene phenotype predictions were when using different reagents types in the same pathogen-infection screen.

The following figure shows the rank-biased overlap (a measure of how similar lists are with regards to top- and bottom-ranked items), when estimating siGENOME off-target knockdown in one of 2 ways:

A) using the maximum TargetScan score for any of the 4 siRNAs in the siGENOME pool

B) using the mean TargetScan score for the 4 siRNAs

If low-complexity pooling increases the degree of off-target effects, we would expect the maximum TargetScan score to produce better model concordance.

And that is what the authors found.  (the two plots show the rank-biased overlap for the top and bottom of the phenotype ranked lists, respectively)

The off-target effect of a 4-siRNA, low-complexity pool is best described by the strongest off-target effect of any of the individual siRNAs.

As discussed in our NAR paper, pooling a minimum of 15 siRNAs is required to reliably prevent off-target effects.

Citations of our Nucleic Acids Research Paper

Citations of our Nucleic Acids Research Paper

Our 2014 Nucleic Acids Research paper provides an excellent overview of the siPOOL technology.  Google Scholar shows that our paper has been cited 64 times.

To put this into perspective, the 2012 PLoS One paper on C911 controls by Buehler et al. has 72 citations.  C911 controls are probably the most effective way to determine whether a single-siRNA phenotype is due to an off-target effect.

These citation numbers show that siPOOLs have good mind share when researchers consider the issue of RNAi off-target effects.

We have noticed, however, that in some cases our NAR paper is cited to justify approaches that we do not endorse.

For example, two recent papers (1, 2) cite our paper as support for the use of Dharmacon ON-TARGETplus 4-siRNA pools to reduce the potential for off-target effects.

Our paper shows, however, that high-complexity siRNA pools (> 15 siRNAs) are needed to reliably reduce off-target effects.

We have also discussed how low-complexity siRNA pools can in fact exacerbate off-target effects.

There’s an old saying that any publicity is good publicity, and we are certainly thankful that these authors have referenced our paper, even if we don’t agree with the interpretations.

And we are especially grateful to all the researchers who have purchased siPOOLs and referred to our products in their publications.

5 factors to consider in multi-gene targeting RNAi screens

5 factors to consider in multi-gene targeting RNAi screens

Summary: Effective functional genomic screening depends on a variety of factors that need to be simultaneously addressed to obtain meaningful results. A recent Cell Reports paper demonstrates this by taking a holistic approach to siRNA screening with the use of multi-isoform/multi-gene targeting to address redundant paralogs and pathways in cancer cells.

The case for multi-gene targeting

Many RNAi screens use arrayed single gene knockdowns to find genes that play an important role in a biological process. The idea is that a single bullet is enough to take down its target leaving a gaping hole that one cannot fail to notice. In some cases, this is true, and is certainly relied upon by drug developers seeking to create specific mono-target drugs.  However, in complex diseases like cancer, cells have evolved fail-safe mechanisms to make them more resistant to external assaults. A single bullet is simply not enough.

Take for example oncogenic protein RAF or Rapidly Accelerated Fibrosarcoma, a tyrosine kinase effector that is a component of the MAPK signalling pathway (Ras-Raf-MEK-ERK). RAF has three isoforms – ARAF, BRAF and RAF1 (also called CRAF). Studies in mouse embryonic development show they all share some form of functional redundancy as knocking out two isoforms produces more severe effects than knocking out each isoform alone.

Screens that target single genes/isoforms therefore tends to bias results towards genes that have no paralogs or only have single isoforms. This was indeed the reason why classical Ras effectors were not identified in previous screens.

Factors to consider in a multi-gene targeting RNAi screen

Determining gene combinations that make sense

The authors of the study did a focussed siRNA screen on 41 RAS effector nodes represented by 84 genes. Out of the 41 nodes, 25 of them had 2-4 functional paralogs where combinatorial gene silencing was carried out with multiple siRNAs. 5 nodes knocked down multiple members of a protein complex. 5 nodes had siRNAs targeting multiple steps within a pathway. Only 6 nodes silenced single genes (highlighted).

Multi-gene targeting screen design

The only caveat with designing such a screen is the requirement for prior knowledge to perform meaningful gene silencing combinations. In this instance, many of the Ras effector pathways are characterized sufficiently to do this well however in other less studied fields, this could be a challenge. Useful tools that would help in designing gene knockdown combinations would include pathway or phenotype databases such as KEGG, REACTOME or Wikipathways. The Phenovault which siTOOLs Biotech is developing, is yet another potentially useful tool.. more details to come!

Number and types of phenotypes

The authors also highlight how a screen that reads only one phenotype might miss other important gene functions. Many RNAi screens sadly still stick to measuring cell proliferation as their only read-out which is greatly influenced by siRNA off-target effects. Here, 5 different phenotypes were measured (cell size, proliferation, apoptosis, reactive oxygen species [ROS], and viability). It was noted that silencing of Cdc42 had little effect on cell viability yet a prominent effect on ROS levels.

To take this up a notch, analysis was also performed at the single-cell level in cells expressing uniform levels of GFP and co-transfected with GFP siRNA. This allowed authors to correlate phenotypes with levels of gene knockdown, generating dose-response curves. How clever!

A lot more work, but adds to data robustness especially when using single siRNAs that are known to be rather variable.

Heterogeneity of cell lines

Many reports and our own observations attest to the heterogenous response of different cell lines to the same treatment. In cancer especially, the large heterogeneity necessitates the use of multiple cell lines. Not doing so would be failing to account for the large genetic diversity observed in the clinic. The authors screened 92 cell lines derived from lung, pancreas and colorectal tissue.

Despite seeing heterogenous responses to node knockdowns, phenotypic responses could be distinguished into  several groups based on effector engagement.  A major group dependended on RAF through direct binding with KRAS, a second major group worked via RSK p90 S6 kinases to drive RSK-mTOR signalling. And a third minor group was dependent on RalGDS. They went on to focus on the first two major groups, naming them KRAS-type and RSK-type respectively.

Reagents – choosing siRNAs and siRNA concentrations

The authors used previously characterized siRNAs to select for more potent siRNAs. This involved an RNAi sensor reporter-based assay that required the generation of 20,000 clones. The reporter was also shRNA-based. Due to heterogeneity in Dicer-mediated cleavage of shRNA, its uncertain if knockdown potency is accurately reflected when translated to siRNAs (read about the difference between shRNAs and siRNAs).

siRNA off-target effects are concentration-dependent

In any case, its a lot of work to characterize all siRNAs to be used in a screen. Furthermore, off-target effects are not addressed.

The authors stuck to a maximal concentration of 12 nM where 2 nM of siRNA was applied per gene. At 2 nM per siRNA, one still risks deregulating other genes. One of the first papers by Aimee Jackson et al., demonstrated an siRNA targeting MAPK14 deregulated many other genes even at concentrations of 1-4 nM.

An important consideration is to ensure total siRNA concentrations are kept constant. In which case, a negative control siRNA has to match or follow the maximal siRNA concentration used. Using different levels of siRNAs runs the risk of biasing off-target effects towards sequences present at higher concentrations.

To learn what the causes, extent and consequences of siRNA off-target effects are, read siTOOLs Technote 1)

Validating results

As with all scientific hypothesis, it helps to arrive at the same conclusion with different approaches.

The two different effector response subgroups identified also responded differently to small molecules. The KRAS-type lines being more sensitive to EGFR and ERK inhibition while the RSK-type lines more sensitive to inhibitors of PDK1, RSK, MTOR, S6K1 and DNA repair enzymes. This was attributed to the latter’s higher basal metabolic activity manifested in larger investments towards oxidative phosphorylation and mitochondrial ribosome maintenance.

By also projecting signatures obtained from cell lines into patient samples (in The Cancer Genome Atlas, TCGA), the subtypes were also effective at predicting differential sensitivity to multiple drug treatments. This highlights the importance in designing effective drug combinations in cancer.

Interestingly, the authors also performed CRISPR pooled screens in parallel. However, due to the restraints of being only able to knockout 1 gene at a time, smaller effects were seen due to gene redundancy. However, they did go on to use CRISPR as well to mutate key genes to affirm the pathway relationships established.

siPOOLs have been used successfully for multi-gene targeting for up to 4 genes, and potentially more. They also safely address off-target effects by high complexity pooling, enabling each siRNA to be applied at picomolar concentrations. For more articles on multi-gene targeting, read an older blogpost:

Understanding gene networks with combinatorial gene knockdown

 

Is it important to avoid microRNA binding sites during siRNA design?

Is it important to avoid microRNA binding sites during siRNA design?

Summary: To address the question of whether one should avoid microRNA binding sites during siRNA design, we examined whether removing siRNAs that share seeds with native microRNAs would reduce the dominance of seed-based off-target effects in RNAi screening.

siRNA design and native microRNA target sites

Recently, we discussed a review of genomics screening strategies.  The authors state:

RNAi screens are powerful and readily implemented discovery tools but suffer from shortcomings arising from their high levels of false negatives and false positives (OTEs) as can be seen when comparing the low concordance among the candidate genes detected in different screens using the same species of virus, e.g., HIV-1, HRV, or IAV (Booker et al., 2011; Bushman et al., 2009; Hao et al., 2013; Perreira et al., 2015; Zhu et al., 2014).

To address these concerns, improvements in the design and synthesis of next-gen RNAi library reagents have been implemented including the elimination of siRNAs with seed sequences that are complementary to microRNA binding sites.

Given that off-target effects via microRNA-like binding are the main source of RNAi screening phenotypes, avoiding native microRNA sites during siRNA design seems like a reasonable strategy.  But does it make much difference in actual RNAi screens?

Hasson et al. 2013 performed a mitophagy screen using the Silencer Select siRNA library.  About 12% of the ~65,000 screened siRNAs have a 7-mer seed shared by a miRBase microRNA.

The screen’s main phenotypic readout, % Parkin translocation (PPT), is strongly affected by seed effects.   The intra-class correlation for siRNAs with the same seed is ~.51 (versus ~.06 for siRNAs with the same target gene).  There appears to be no difference between how siRNAs with or without microRNA seeds behave:

Is it important to avoid microRNA binding sites during siRNA design?

The same thing is found if we look at a less specific phenotype like cell count (which should be more broadly susceptible to off-target effects, as more genes should affect this phenotype):

Is it important to avoid microRNA binding sites during siRNA design?

And if we look at seeds that are enriched at the top of the screening list (sorted by descending PPT), we also don’t see much difference between siRNAs with or without native microRNA seeds.  (Note that the seed p-value is calculated in a similar way to RSA, based on how over-represented a seed is towards the top of a ranked list)

Is it important to avoid microRNA binding sites during siRNA design?

We also examined a general phenotypic readout (cell viability) in a dozen large-scale RNAi screens.

For some screens, we do see a slight shift in the values for siRNAs with or without native microRNA seeds.

For example, a genome-wide screen of Panda et al. 2017 (also using the Silencer Select library) shows a slight decrease in viability for siRNAs with native microRNA seeds:

Is it important to avoid microRNA binding sites during siRNA design?

Removing those siRNAs does not change the dominance of seed-base off-targets.

The intra-class correlation (ICC) for siRNAs with the same 7-mer seed is ~.53, with or without the inclusion of siRNAs with native microRNA seeds, while ICC for siRNAs with the same target gene is only  ~.06.

Coming back to the quote from the review article on genomic screening, next-gen RNAi library reagents that avoid native microRNA seeds are not expected to be much better than siRNAs that include them.

The most effective way to avoid seed-based off-target effects is to use high-complexity siRNA pools (siPOOLs). Learn more about siPOOLs

 

Correcting seed-based off-target effects in RNAi screens

Correcting seed-based off-target effects in RNAi screens

Summary: Correcting for seed-based off-targets can improve the results from RNAi screening.  However, the correlation between siRNAs for the same gene is still poor and the strongest screening hits remain difficult to interpret.

Seed-based off-target correction has little effect on reagent reproducibility

Given that seed-based off-targets are the main cause of phenotypes in RNAi screening, trying to correct for those effects makes good sense.

The dominance of seed-based off-targets means that independent siRNAs for the same gene usually show poor correlation.

If one could correct for the seed effect, the correlation between siRNAs targeting the same gene may improve.

One straightforward way to do seed correction is to subtract the ‘seed median’ from each siRNA.  (The seed median is the median for all siRNAs having the given seed.)

This was the approach used by Grohar et al. in a recent genome-wide survey of EWS-FLI1 splicing (involved in Ewing sarcoma).  They used the Silencer Select library, which has 3 siRNAs per target gene.

After seed correction, there is only minor improvement in the correlation between siRNAs targeting the same gene.  The intra-class correlation (ICC) improves from 0.031 to 0.037.  The ICC for siRNAs with the same 7-mer seed decreases from 0.576 to 0.261.

Although we have reduced the seed-based signal, it has not resulted in a correspondingly large improvement in the gene-based signal.

More sophisticated seed correction can improve reagent correlation

Grohar et al. used a simple seed-median subtraction method to correct their screening results.

A more sophisticated method (scsR) was developed by Franceshini et al. for seed-based correction of screening data.  It corrects using the mean value for siRNAs with the same seed, and weighs the correction using the standard deviation the values.  This allows seeds with a more consistent effect to contribute more to the data normalisation.

Applying the scsR method to the Grohar data, ICC for siRNAs targeting the same gene increases from 0.031 to 0.041.  It is better than the increase with seed-median subtraction (0.037), but is still only a fairly minor improvement (plot created using random selection of 10,000 pairs of siRNAs that target the same gene):

 

Off-target correction increases double-hit rate in top siRNAs of RNAi screen

The following plot shows the count for single-hit and double-hit genes as we go through the top 1000 siRNAs (of ~60K screened in total).  Double-hit means that the gene is covered by 2 (or more) hit siRNAs.

Despite the small improvement in reagent correlation, the double-hit rate is essentially the same using simple seed-median subtraction or the more advanced scsR method.

Furthermore, the number of double-hits is higher than what we’d expect by chance.

This shows that, despite the noise from off-target effects, there is some on-target signal that can be detected.

siRNAs with the strongest phenotypes remain difficult to interpret

Despite the fact that the double-hit count is higher than expected by change, most of the genes targeted by the strongest siRNAs are single-hits.  siRNAs with the strongest phenotypes remain difficult to interpret.

Seed correction is best suited for single-siRNA libraries.  Low-complexity pools, like siGENOME or ON-TARGETplus, are less amenable to effective seed correction since there are (usually) 4 different seeds per pool.  This reduces the effectiveness of seed-based correction, even though seed-based off-target effects remain the primary determinant of observed phenotypes (as discussed here, here , and here).

The best way to correct for seed-based off-targets is to avoid them in the first place.  Using more specific reagents, like high-complexity siPOOLs, is the key to generating interpretable RNAi screening results.

For help with seed correction or other RNAi screening data analysis with the Phenovault, contact us at info@sitools.de

Little correlation between Dharmacon siGENOME and ON-TARGETplus reagents

Little correlation between Dharmacon siGENOME and ON-TARGETplus reagents

The most common way to validate hits from Dharmacon siGENOME screens is to test the individual siRNAs from candidate pool hits (siGENOME reagents are low-complexity pools of 4 siRNAs).  In this deconvolution round, we normally see that the individual siRNAs for genes behave very differently and seed effects dominate (discussed here and here).

One could argue that deconvolution is not the correct way to validate candidate hits (even though it’s the method recommended by Dharmacon),  as testing the siRNAs individually will result in seed effects that are suppressed when the siRNAs are pooled.  One problem with this argument is that low-complexity pooling does not get rid of off-target effects (e.g. Fig 5 in this paper), something that is better done via high-complexity pooling.  But assuming it were true, validating with a second Dharmacon pool would be better.

Tejedor et al. (2015) performed a genome-wide Dharmacon siGENOME screen for regulators of Fas/CD95 alternative splicing.  ~1500 genes were identified by a deep-sequencing approach.  ~400 of those were confirmed by high-throughput capillary electrophoresis (HTCE, LabChip).  They then retested those ~400 genes (again by HTCE) using Dharmacon ON-TARGETplus pools.

The following plot shows the values for the siGENOME and ON-TARGETplus pools for the same genes (i.e. each point corresponds to 1 gene).

What’s measured is the percent of splice variants that include exon 6 following siRNA treatment.  That was compared to the values for a plate negative control (untransfected wells) and converted to a robust Z-score.  This is the main readout from the paper.

The Pearson correlation improves if the strong outlier at -150 for siGENOME is removed (R = 0.25), while the Spearman correlation is unchanged.

We see that a fairly small number of genes are giving reproducibly strong phenotypes (e.g. 13 of 400 have robust Z-scores less than -15 for both siGENOME and ON-TARGETplus reagents).

If we remove those 13 strong hit genes, the correlation approaches zero:

Even if the strong outlier for siGENOME is removed, the correlation is still near zero:

Although using a second Dharmacon pool removes some of the arbitrariness of defining validated hits (e.g. saying that 3 of 4 siRNAs must exceed a Z-score cut-off of X, or 2 of 4 siRNAs must exceed a Z-score cut-off of Y), the end result is similar:  A few strong  genes show reproducible phenotypes, while many of the strongest screening hits show inconsistent results.  The main problem, off-target effects in the main screen, is not fixed.

postscript

Tejedor et al. say that 200 genes were confirmed by ON-TARGETplus validation.  They consider a gene confirmed if the absolute value of the robust Z-score is greater than 2.  The Z-score is calculated using the median for untransfected plate controls.  I suspect that a significant proportion of randomly selected genes would also have passed this cut-off.

In table S3 (which has the ON-TARGETplus validation results), there are actually only 177 genes (including 2 controls) that meet this cutoff.  The supplementary methods state: Genes for which Z was >2 or <-2 were considered as positive, and a total number of 200 genes were finally selected as high confidence hits.

Which suggests that genes outside the cut-off were chosen to bring the number up to 200.

But if we look at the Excel sheet with the ‘200 hit genes’, it has 200 rows, but only 199 genes.  The header was included in the count.

This type of off-by-one error is probably not that uncommon.  In a case like this, it does not matter so much.

One case where it did matter was in the Duke/Potti scandal.  The forensic bioinformatics work of the heroes of the Duke scandal found that, when trying to reproduce the results from published software, one of the input files caused problems because of an off-by-one error created by a column header.  That was one of many difficulties in reproducing the Potti paper’s results which eventually led to its exposure.

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