We are very excited about the results which have been posted in Eterna:
and the data can be found in
Google spreadsheets: Eterna R100 [A]/[B]
or the Excel file: Eterna R100 [A]/[B]
R2 (2 State model)
The R2 lab winners made by JR showed the overall same tendency.
Sequence B came before A
The MS2 was sharing lane with sequence B
MS2 in between the oligos
JR design, Score 97%
The general trend was having the MS2 in between the oligo complements and using the main part of the design up. This is similar to what we have seen in the logic gate labs already.
R2 Drawings overview
The more unusual bit in the majority solves, are that the B sequence were doing a crossover pairing up (which is more usual in microRNA turnoff labs) in the majority of the winners. With single inputs that is.
R3 (3 State model)
Here the jandersonlee winners had extensive sequence sharing between both sequence A and B but also B and MS2.
A sequence before B
A and B sharing lanes - or really magnet segment :)
B and MS2 sharing lanes
MS2 on the outside - not between oligo complements
jandersonlee winner, score 99%
Here the main trend was using only part of the design sequence and having the rest of the excess bases tucked away as static stems.
R3 drawings overview
Perspective on R2 versus R3
The R3 winners follow a true blooded exclusion solving style. By the choices made, they make sure that the unwanted sequence/MS2 is either included or excluded.
When I say exclusion solving style I mean a solving strategy where the switch is made by sequences sharing lanes and where one will actively exclude the other by binding and vice versa.
The exclusion style as followed in R3, is a short cut way to achieve a switch. It is the solving style that is simplest, requires least space and involves fewest switching pairs.
This strategy has been extremely successful in the microRNA-in, reporter-out labs already. However these labs did not have to have the MS2 to fit in the equation too. Which I think is what complicates things, when comparing the reporter labs with the R2 and R3 labs.
The exclusion style takes extensive sequence sharing. And from both sides of the sequences - no matter which order the oligos are placed in relation to each other. Plus the same goes for sequence sharing in relation to MS2 too. Which again takes all the sequences being a match for this in the first place.
I did make an exclusion style series of designs in R2, where A and B shared lane, but none of them scored well. (With a max of 65%)
So if the overlaps between sequences are not good enough - then the best road to get to a solve, seem to be the one that involves going more complex and using a far bigger part of the RNA sequence to solve the problem. By placing the MS2 a middle spot and to place some of the switch elements further apart, to give space to put sequences in between for doing the switch job. To either turn on or off MS2 or sequence inputs.
Instead of having the switch elements themselves do the job directly on each other. As is done with pure exclusion style.
The R2 winners do something kind of in between exclusion style and having sequences in between the switch elements do the job. They still use the fact that MS2 and sequence B can be made overlap and share sequence. And as such get half the benefit from the exclusion style, plus escapes its downfall, due to sequences not matching well enough for a successful overlap, when the oligos are reversed. R2 is oligo B comes before A where R3 is oligo A comes before B.
The morale of this story is that it is easier to get two sequences (switch elements) to share 1 overlap nicely for taking turns, than to make 3 sequences (switch elements) share 2 overlaps evenly between them and take turns. The R3 results show that the latter is possible, but the R2 results underlines that it seems to be harder.
It seems - no surprise - that the exclusion style strategy is absolutely strongest - but only when the sequences are able to overlap perfectly with each other for achieving exclusion.
I have made a pattern sum up for the single input labs and the labs with predefined sequence placement. I have been interested in what main patterns turn up among the winners and top scoring designs in these labs.
Basically this is a hint for what I think will work for next round.
Sensor A MS2 off
The top scoring designs follows this pattern.
A before MS2
A at bad spot (5’ side) - easier for kickoff
Design complex at 5’ end
Inspired by Omei
Sensor A MS2 ON
MS2 before A
A sequence at best turn on spot at 3’ side
Design complex at 3’ side
These trends are overall followed for all the winners.
Score 100, design by Brourd
The designs that do absolutely best for fold change, keeps a magnet G dangle at the tail. This do even better than a similar magnet G dangle at gap position.
My designs scoring 100% also keeps this magnet dangle, but have a different MS2 turnoff. One I transported from the jandersonlee’s winner in the microRNA 208a lab. Was real nice to see it work here also.
My MS2 turnoff is more bendy, something that can be a problem for a switching area. Brourd’s MS2 turnoff is almost unnatural straight in the second state. However I would love to see this straight MS2 turnoff put onto other designs, to see if it still gives raise to extra high fold changes.
I think we have a very good shot at moving elements onto other designs as long as they are not supposed to directly interact with the new design they are moved into. A MS2 and its turnoff can regularly be individual self sufficient systems as long as they are in 1 and 2 input systems.
Sensor B MS2 OFF
Main type among the winners.
B before MS2
B at bad spot (5’ side) - easier for kickoff
Design complex at 5’ end position
As the Sensor B MS2 ON lab, this lab with the strong B sequence shows bigger tolerance than usual.
A good deal of the winning designs have very bending binds between design and sequence B.
JL mod by mat, score 94%
Just like the Sensor B MS2 ON lab, this lab has high fold change error rates. And also some surprisingly low cluster counts in one of the states.
Now I wonder if there is a relation between the designs having these strong bending pairing between sequence B and the RNA design. And there really seem to be. The top 2-5 has low cluster counts and high fold change error rates. And these designs are siblings of the above shown design.
Sensor B MS2 ON
This lab has a tendency for reversal of the sequence landing spots. The B sequence is a rather strong one. And as such I suspect this effect the equation. The strong sequence has the ability to also hook up at a less optimal place - early in the sequence, where the much weaker A sequence is more dependent on getting the optimal spot late in the RNA design.
Notice that Lroppy’s winner do not have the B sequence bound up at the furthest 3’ end - which is generally the best site to place a sequence one wants to catch and turn on. Because this sequence is strong, so it doesn’t need it.
Design by Lroppy, score 98%
Other winning variant, of more like the Sensor A MS2 ON pattern.
So this lab has high scoring designs that do not all follow the same path of the Sensor A MS2 on lab as neatly as the design above. Where MS2 would have been followed by the B sequence, last in the design at the 3’ end.) Instead some of the following patterns pops up in high scorers. (Along with more)
Reversal of the sequence binding spot, so sequence B comes before MS2
A static stem in between MS2 and B. (With MS2 first and sequence B last, although also sometimes reversed)
The solve can pop up further towards the 5’ end - which is generally worse. (Except for the miRNA-in, reporter-out lab, round 1 - for whatever reason. The only thing that makes it stand out from the round 2, is that it has a real short oligo involved.)
Though there are more of these among the designs winner designs with reversed binding site pattern that have a higher fold change error rate. As I should have mentioned earlier, I’m generally using a cut off line in the data on a Fold change error rate of 1.15. I have been using this cutoff line lately. Most of the data is good enough to leave enough winners back afterwards.
This lab in particular has a higher fold change error rate than the others. Not sure why.
[A]/[B] with predefined binding site
The high scorers follows this pattern.
B before A
MS2 turns up at end position
Malcolm's JR mod
The related but unconstrained lab (R2) has its the MS2 positioned in the middle in the winning designs.
In comparison the lab with predefined binding site forces the MS2 to shift place to the end of the sequence. No design scoring over 73%, have their MS2 placed in between the oligos.
Thereby the lab looses its option of having the MS2 and the B sequence sharing lane (or really rather magnet segment) as was the case in the majority of the R2 winners. The constrained lab did achieve a similar scoring winner. But it seems it was a bit harder making winners in the constrained lab. Time will tell.
However it was yet again confirmed that MS2 can be happy at ends of the sequence - at least as long as there two sequence inputs. Having MS2 at ends were generally less optimal in single input labs and Riboswitch on a chip labs. There it generally helped having MS2 held from both ends to have a chance to get it turned off. But when MS2 are having similar length playmates, it becomes more willing to take the odd position and still fold open and be turned off - even if not being held from both ends.
[A]/[B] with predefined binding site (Alternative)
A before B
MS2 at middle position
Salish mod of JR
This is absolutely opposite to what would happen had the binding sites not been predefined. The weaker TB A gets the worst spot for turn on. See the R2 overview:
While Salish solve shows it is possible solving the other way too. The predefined lab, forces reversal of the pattern that generally pops up in R2 winners. But it seems to be just as solvable this way. The global folds are even better for the predefined lab.
It seems that when two inputs are involved, they are a bit less picky about their landing spot, compared to when single inputs are involved.
I think when two input gets involved and one have a MS2 in the middle and the oligos far apart, the sequences put in between gets to play a stronger role, one that also allows for reversal if things are balanced right.
I however still think one particular way of solving will be better than the other. And as for now I’m betting on the unconstrained one.
GRAMMAR OF THE RNA
Single input rules
Want a single input lab turned on? - Place the sequence at the best spot (3’ side) in state 2. Place MS2 before. (Example labs: Sensor A MS2 on, microRNA 208a)
Want a single input lab turned off? - Place the sequence at the bad spot (5’ side) in state 1. Place MS2 after. (Example lab: Sensor A MS2 off, Sensor B MS2 off)
Alternative turnoff route is to give the MS2 a turnoff sequence and split the pairing of the oligo on both sides of the MS2. (Sensor v3 - variant 1 and variant 2)
Sum up: The late bit in the RNA sequence (3’ end) is best for whatever one wants turned on. The early bit in the RNA sequence (5’ end) is best for whatever one wants turned off.
Only if the oligo is real strong like the B one, it can shift balance and side. Then it can sometimes take the bad spot or less optimal spots and still get turned on.
Double input rules - Exclusion labs
If possible, let the sequences overlap/share lanes. This is a quick and dirty way of achieving an exclusion switch, where one sequence kicks out the other. If not possible, place the MS2 in the middle and the oligos to the sides.
Place leaving sequence at the bad spot (3’) end and the staying sequence at the best spot at 5’ end. (Which doesn’t have to be exactly at the 3’ end in the RNA design always - it's just the oligo you want to stay that has to be 3’ in relation to the oligo you want kicked out.)
(Example labs: R2, R3, miRNA-in, reporter-out round 1 and 2)
Basically this is just the two options in the single input rules above, added together.
However as a twist the predefined labs showed that other patterns would pop up, that were working too, when binding site was forced. In the alternative binding site lab, the reversal of the above mentioned pattern seemed to work just as well. But I suspect that one will turn out better than the other.
Exclusion labs without MS2
If possible, let the sequences overlap/share lanes. Put the oligo you want turned off before, or 5’ end of the oligo you want turned on.
It seems the miRNA-in, reporter-out with short reporter, round 1, preferred to be at the bad spot at the 5’ end while the miRNA-in, reporter-out with long reporter, round 2, preferred the better 3’ spot. (Example labs miRNA-in, reporter-out round 1 and 2). Something I don’t get why yet.
What sequence strength may reveal
A thing I count worth looking out for.
The sequence B is really strong - it has lots of strong bases.
I already noticed that the single input B sequence labs alone got skewed from the regular pattern that pops up in the pure Sequence A labs and that I was expecting. I think we can actually use this for something.
I strongly suspect that we can use the strong sequence B to investigate which crazy solves each lab will tolerate - also for the A and B mixed labs - where the weaker sequence A when alone, will highlight what areas are bad for a solve, by gleaming by its absence and extremely bad scores.
This Sensor B MS2 ON lab had a lot of crazy possible solves among the near winners.
( I messed up the round number in the title sorry)
This data can be used to assist in designing for the next round. It contains report files on the secondary structures like how many of the same structures are used as well as markers for those score ranges. I also included the raw DPAT data.
I was thinking. If you use the marker data use #Markers as one of your hashtags please and #PartitionFunction if you use the partition function values, and #PairingProbs if you use pairing prob data..
What I wish to convey to you, is the image of shifting balance.
Equal sized playmates and the game can begin. Each taking terms of being up and down.
In our switch RNA designs, the teeter totter is unevenly adjusted from the start.
Not each seat is equally hospitable. Not each playmate being equally strong. One sequence has much weaker base content than the other. To achieve balance and a good game, things needs to be adjusted.
Balanced seesaw between playmates. Both are going to get an enjoyable ride.
When the bigger sibling moves toward the middle, balance can be achieved and the smaller sibling will not get a too bumpy or flying ride. Now most seesaws are not adjustable like the one above.
TB A sequence with weak bases is the little brother sequence, needing the better spot on the teeter tooter.
The teeter totter being the RNA sequence. The oligos being the kids. MS2, a reporter sequence or a static stem can be the wooden stem.
In case of two RNA inputs, MS2 will tend to take the middle spot, unless things needs to get really skewed - like in the OR lab. What to place where, will also depend on what you want done.
Single and double input labs
As I mentioned earlier:
The late bit in the RNA sequence (3’ end) is best for whatever one wants turned on. The early bit in the RNA sequence (5’ end) is best for whatever one wants turned off.
While this happens for single input labs, this seems to be what is naturally inclined to happen, even in double input switches where one sequence needs to leave. This be both if they are sharing lanes or being apart.
Getting the balance right
However it is very well possible reversing the order of the oligos and place the weak oligo at the seemingly worse spot. As was done in the one lab with predefined binding side: See the section: [A]/[B] with predefined binding site (Alternative)
It just takes adjustments of some of the other levers, to shift balance between the sequences and the binding spots. Just to mention a few.
Strength of the individual RNA sequence matter
Which end the design complex is placed, matter
What end the oligo is placed at, matter
A complement prolongment can shift balance
A MS2 turnoff sequence can change balance - short distance turnoff
Which side a MS2 is turned of at, can shift balance
A tail magnet can shift balance - long distance turnoff
A static stem in the switching area can shift balance
The placement of MS2 inside the RNA sequence matter - not equally effective at ends
And so on
The concentration of the oligos are the switch motor, their concentration a driving force for switching.
Oligo strength and concentration will determine where they will be most likely succeeding in landing and most likely leaving
Extra sequence in between switching elements in for turnoffs
Lane sharing - oligo concentration either welcome or force oligo out
MS2 used as turnoff for oligos
Example of shifting balance
I have shared an example of shifting balance. In the R2 lab it was not possible for me to make the sequences overlap at the order I wanted them in - the leaving sequence before the staying sequence and still have them share lanes. Instead I did something else to shift the balance.
Related post: Grammar of the RNA
I have been wondering about what decides at what end the design complex turns up. I have generally wanted to put them at 3’ end, after seeing the initial success from the microRNA 208a lab.
However this turned out to be not the full truth. I think there is something else that determines what end the design complex ends at.
The A/B designs with A and B sequence turnoff, tended to have their design complex at the ‘5 end of the sequence instead.
Basically the single input ON and OFF labs, the ON labs have had the design complex at the 3’ end and the OFF designs have had the design complex at the ‘5 end. Each choosing the position that sent the MS2 towards the middle of the design.
I think the R3 winners were moved towards the 5’ end, because the design complex had its MS2 last. Had the design complex been moved to the 3’ end, the MS2 would have been put at the absolute end of the design, which it has generally shown to work worse - although it is more possible in designs with two inputs compared to one - something which is also shown in the [A]/[B] with predefined binding site lab.
If MS2 is at one end in the design complex and not between oligos, then the design complex seems to choose whatever side that gives the MS2 a more middle position over an end position.
The pattern with the design complex being skewed to one end of the design, typically happens when sequences can be made share lane. This result in much smaller area of the design, where designs that can not be made sharing their switching elements, tend to take up far more of the available space.
Lab quest: Which end is the best position in the RNA design
Anyway I suggest and am planning myself to moving some of last round successful designs in open labs, to the opposite end of the RNA sequence to see if they have any particular bias in relation to placement - which I strongly suspect they will.
I mark these with the hashtag: #Design complex moved to 5’ end or #Design complex moved to 3’ end.
Especially the winners in the two miRNA-in, reporter-out labs, round 1 and 2 have been driving me nuts. Each has its winners turn up at either end of the RNA sequence. What I can't understand is why they don't both turn up at the same end.
Majority winner type from each lab:
Now these labs don’t have MS2’s. It can be that they are not so fussy about their placements, as have they had a MS2.
The only real difference between these labs, are that the reporter (bound in state 1) is short in round 1, but longer in round 2. Ok, the TB signatures are different also. But not by much. They are in same mod of colors/bases.
In the Single input A and B labs, the position of the MS2 seemed to be involved in determining what end the design complex ended at. Can it be that the different reporters - in MS2's place - are whats destining these design complex to each their end of the RNA sequence?
The only thing I can see they accomplish by their exact position of the design complex is to get their strongest base section put in place for a good dangling end position.
I did experiments to first round designs with already known results, as I expected that it would matter which end a design complex were placed at. 5’ prime or 3’. The results so far seems to confirm.
I took jandersonlee’s 3 round 1 winners that had the design complex placed at the 5’ end and slided the design complex to the 3’ end.
All slided designs resulted in worse scores (in the 60 and 70’ties).
The low scoring 3’ positioned ones have less negative kcal in both vienna and Nupack compared to the original winning 5’ positioned ones.
More negative kcal seems to be a tell tale sign for which end is the better position for the design complex, particular in those cases where the inputs share landing spot.
MS2 positioning in the sequence
Most of these switch labs prefer a somewhat middle placement in the sequence of their MS2.
R2 - All the winning designs have their MS2 in the middle or late middle of the sequence.
R3 - All the winning design have their MS2 in the middle of the sequence. A few - early middle.
Sensor A MS2 off - MS2 in early middle
Sensor A MS2 on - MS2 in middle, late middle and a few early middle.
Sensor B MS2 off - Early middle, middle + a few late middle. As first round started to indicate, the stronger B input has more choice where to bind up with the sequence and still get a good score.
Sensor B MS2 on - Middle placed, late middle + a few early middle
[A]/[B] with predefined binding site winners is the exception and holds the MS2 at 3’ end. However it doesn’t yield as many winners as the other labs.
[A]/[B] with predefined binding site (alternative) - MS2 in middle or late middle.
Positioning of inputs
The trends for positioning of the inputs that started showing in first round continues for this round. The input positions show the same pattern as I mentioned in the last section in this earlier post.
I set a minimum cluster count at 50. And looked at the sequences for designs scoring 94% and above.
Sensor A MS2 ON
Winners generally have the A input at 3’ end.
Sensor B MS2 ON
Trend towards having B input at 3’ end, but mixed pattern shows as well. The strong B input can be placed in the middle or at either end and work just fine. Just as in first round.
One of the designs from first round, tolerated a total reversal of the design complex with raised score from 96% to 100%. B input ended at the 5’ end. Which is opposite of what I would have expected had the this lab had input A instead.
Sensor A MS2 OFF
Winners generally have A input at 3’ end.
Sensor B MS2 OFF
Winners mostly has the B input placed at 5’ end.
Input position sum up
As from the above summary, the OFF designs tend to follow same structure pattern for placement of inputs and so do the ON designs. However the different strength of the two inputs seems to be enough to throw this structure pattern a bit off sometimes.
So seemingly something as simple as variation of the input sequence and its strength opens up for different positioning of the input in relation to the design sequence. The stronger B input that holds more strong bases - and thus have better options for a strong pairing - can bind more places in relation to the design sequence and legally.
However on the other hand it seems much easier making winning designs with the weaker A input, no matter if it is a MS2 turnon or turnoff lab.
Most of the designs where I put the design complex at opposite end of the design, didn’t benefit. Which kind of makes sense as the MS2 has a favorite position in relation to the design - middle mostly and the input also has its favorite position too - in relation to if it is an on or off lab. Move the design complex and you touches both MS2 position and input position at once.
[A]/[B] with predefined binding site labs
I have been claiming that A should be coming before B, or rather that the sequence that is present in state 1 but needs to leave in state 2 the leaving input should be before the staying input. And I think it is particularly effective if the inputs overlap directly. However I have been on the watchout for if there should also be something to it for inputs that were placed more apart.
For background, see section: The "A before B" rule
First round results clearly showed that it was absolutely possible having the leaving sequence last and the staying first. [A]/[B] with predefined binding site (alternative)
For background post, see section: Reversed sequence pattern
While this still holds true for round 2, a more clear pattern now shows, where the lab that has the leaving input first and the staying input last, gets the most of the winners. ([A]/[B] with predefined binding site) Something I find quite interesting.
Perspective on input order
One thing else I’m interested in knowing is how much the individual sequence of the inputs and its strength has to do with the order of staying and leaving input also. Would the exact same pattern show, if input the inputs were opposite in strength?
There never seams to be a single thing that eliminates a design. There are however many trends and RNA does tend to want to follow a "trend" but only insomuch as it feels like it. It follows the rule until it doesnt but generally stays true. Looking at plots of Ensemble Diversity for the FMN and the Oligo Round 1 I found that a value of 10 or less for Exclusion/Off labs and 15 or less for Same State/On labs had the vast majority of high scoring designs with maybe a couple outliers occasionally. I would plot Round 2 but the scores are not accessible through the Eterna UI yet for DPAT to crunch.
I will go through designs solved in different styles for R2 and R3 and sum up what I think characterises them.
What causes high global fold change
Last round I mentioned that there seemed to be a relation between high global fold change and designs with direct overlapping inputs (MS2 included) and/or static stem in the switching area.
That relation seems to continue.
Static stem in the switching area + lane sharing
(The numbers in this post are found at cluster count at minimum 50, and score minimum 94%, with exception for this section)
In last round, the design with highest global fold change in R2 was my 92% static stemmed and partial input overlapping design. Note: The rerun of the original design didn’t score 92% as in round 1, but only 82% when rerun. Mat and jandersonlee managed to make winners out of it
This time I could pretty much pick out the mutants of it (6348262), just by sorting the designs after high global fold change. :)
Global fold change range up till 10%
Fold change up till 12%
Staying input after leaving input (opposite to preferred order)
Static stem in the switching area
Partial lane sharing
Partial moving switch
There were however very few winners in this style. I also think this has to do with that the R2 lab can’t have the leaving input before the staying input while also overlapping well. Something which is possible in the R3 design, that despite having one more input - something that usually complicates things more, yielded far more winners. This lab also had the highest of all global fold changes - both in first round, but especially in this round.
Maximum overlap between the inputs and reporter
There was one notable exception among the designs with high global fold change that took a different road. The design 6 3 by dl2007. I think this one holds a bunch of winners in it if mutated a bit.
Global fold change range up till 8%
Fold change up till 8%
Staying input after leaving input (opposite to wanted order)
Lane sharing between all inputs
Partial moving switch
This design follows the exact same approach as jandersonlee’s R3 winners that went for maximum overlap between the all the inputs. This design also gets a fine global fold change.
R3, score 100%, fold change 16.44, global fold change 12.71
Image taken from this post + added a few missing pink lines.
R2 - Inputs placed apart style
The approach that won out for the R2 lab round 2 as the majority type, was distancing the inputs and going more full moving. This created a load of winners with the highest local fold change reached. Although they only reached a max global fold change of 3 for now.
This design type mainly hit through in a bunch of JR designs + mutants. Here is one with partial lane sharing. Worth noting is that by adding distance between the inputs, JR actually puts the leaving input before the staying input.
Global fold change range up till 3%
Fold change up till 23%
Leaving input before staying input (preferred order of the inputs)
Partial lane sharing/No lane sharing - depending on which JR design and mutant
Static stem in switching area in some cases/none in others
Full moving switch/partial moving switch
R3 - Inputs placed apart style
While most of the R3 winners were derivatives of jandersonlee’s round 1 winners, skyblue demonstrated that R3 can be solved with its inputs placed apart too.
Global fold change less than 1
Fold change up to 17%
Inputs non overlapping
Full moving switch
These solves gets high fold change but very low global fold change. There are very few of these among the winners.
R3 - Total input overlap - and inputs in good order
The lab with overall best global fold change was again the R3 one. Main part of the winners were of the jandersonlee type that also hit through as winners 1 round.
Global fold change range up till 34%
Fold change up to 37%
Lane sharing by sequence overlap between switch elements.
Leaving input before staying input (preferred order)
Global fold change
Global fold change overview
For now it still seems that that specific things in a design makes it easier to achieve a high global fold change. Placed in order from more important to less important. The last option can be used to circumvent that one hasn’t gotten the inputs in preferred order.
Extensive overlapping between inputs
Inputs in preferred order
A static stem in the switching area
High fold change versus global fold change
I have been thinking about the relation between high local fold change and global fold change.
It is possible reaching a very high fold change with a high degree of freedom for switching (towards full moving and away from static) But I think it comes at a cost of global fold change and the switch being harder to control.
Just like for the FMN/MS2 switches, the designs that reached highest fold change, allowed an extra degree of switch freedom (only neck static but with static stem in the switching area removed) compared to the more partial moving switches (having 1 or more static stems besides the neck). But I suspect these less partial moving switches may be harder to make switch in a very precise way at a larger range of concentrations.
The strictly overlapping R3 designs can be likened with the partial moving switches. By the extensive overlapping their degree of freedom is limited.
One see far higher fold changes in the TB switches with fewer inputs. The more inputs, the more limited fold change? Likely, because more inputs also means far fewer degrees of freedom.
The overlapping style is sweet and effective when there is a good match between the input/reporter, but have a very picky sweet spot for making winners. Especially if the inputs are not able to overlap in their seemingly preferred order. (Leaving before staying)
There are several ways to go, to escape the seeming penalty of non preferred ordered inputs
It seems that moving the inputs apart and go more towards a bigger switching area is the way to go to reach a high fold change and lots of winners. As in JR’s R2 winners.
If the goal is high global fold change is the goal, and the inputs were not in preferred order, but it could be made work none the less, by adding a switching stem in the switching area and partial overlap,. As in my static stem in the switching area design type.
Similar dl2007’s R2 design with extensive overlapping of inputs also achieved a fine global fold change despite the inputs also being in non preferred order.
But when inputs fits each other for a good ordered overlap that will be my preferred method. For both high local and global fold change.