The H3K79me2 eviction gradient is not a ratio of sums dominated by a few bins: the MEDIAN per-bin log2 share ratio is itself monotone at Spearman -1.000, the fraction of bins individually below 1 falls 89.3% to 24.7%, the bootstrap span interval is [-0.651, -0.545], and no single bin moves the span by more than 0.0057
also called Q130 DEVIATION 1 · the per-bin check · the median bin agrees with the sum
The headline number adds up signal across three hundred DNA stretches at a time, so it could in principle be set by one or two unusual ones. Checking the typical stretch instead of the total gives the same answer, and removing the single most influential stretch barely changes it.
The same thing, for a scientist
**The statistic at issue.** `R = (sum over the quartile of the drug track / its genome total) / (the same for DMSO)` is a ratio of SUMS. Q124's own integrity check - *no track is one-artefact-dominated* - is a WHOLE-TRACK check whose worst value was 0.00013 of the non-blacklisted total, and it says nothing about whether one bin inside a 307-bin quartile carries that quartile's ratio [E1]. **Four checks, all POST HOC and none in the pre-registration** [E2]: | | Q1 | Q2 | Q3 | Q4 | |---|---:|---:|---:|---:| | R, the ratio of sums | 0.5860 | 0.8188 | 0.9280 | 1.1865 | | **median per-bin log2 share ratio** | **-0.7213** | -0.1680 | -0.0158 | **+0.2673** | | the same as a fold change | 0.607 | 0.890 | 0.989 | 1.204 | | fraction of bins individually below 1 | 0.893 | 0.650 | 0.511 | **0.247** | | largest single bin's share of the quartile's DMSO signal | 0.0066 | 0.0084 | 0.0110 | 0.0111 | - **The median per-bin ratio is monotone at Spearman -1.000**, and a median cannot be moved by one bin however large. - **The largest single bin holds between 0.66% and 1.11% of its quartile's DMSO signal**, so no quartile has a dominant bin. - **Bin-level bootstrap, 2,000 resamples, seed 20260923**: the Q1-minus-Q4 span has a 95% interval of **[-0.651, -0.545]**, excluding zero, and **2,000 of 2,000 resamples keep the observed sign**. - **Leave-one-bin-out**: dropping the single most influential bin from Q1 or from Q4 changes the span by at most **0.0057**, against an observed span of 0.6005 - about one part in a hundred. **And the same battery run on the DOT1L-inhibitor arm returns the opposite verdict**, which is what makes it an instrument and not a rubber stamp: under SGC0946 the median per-bin ratio is not monotone (Spearman -0.400), the bootstrap span interval is [-0.0078, +0.0138] and **spans zero**, and only 73.1% of resamples keep the observed sign [E3].
What a bin-level bootstrap does and does not measure
It resamples bins within one library and answers: how much of the span is attributable to which bins happened to be in each quartile? That is the question the ratio-of-sums concern raises, and it is answered.
It says nothing about library-to-library variation, which is the variation a p-value on a biological claim would need, and which is unmeasurable in this deposit because GSE260742 contains one H3K79me2 ChIP per genotype per drug. The only estimate of that quantity anywhere in this series is Q125's two H2AK119ub conventions of one culture, whose spans differ by 2.4-fold while agreeing in sign. A reader should treat [-0.651, -0.545] as narrow for the wrong reason - it is narrow because 307 bins is a lot of bins, not because the measurement is reproducible.
| script | analyses/q130-does-the-eviction-gradient-exist-in-h3k79me2/deviation1_per_bin_robustness.py |
|---|---|
| status | run |
| result summary | Median per-bin log2 ratio monotone at Spearman -1.000; largest bin 0.66-1.11% of its quartile; bootstrap span CI [-0.651, -0.545] with 100% sign agreement; leave-one-out worst change 0.0057. |
| seed | 20260923 |
| n bootstrap | 2000 |
| absence class | measured-not-analysed |
| absence class note | No per-bin robustness check of any eviction-quartile statistic existed in this corpus before this beat; Q125's gradient was reported as a ratio of sums with a matched null and no per-bin statistic. The same check could and should be run retrospectively on Q125's three marks. |
| absence class assigned | 2026-09-23 (Q130) |
| epigenetic readout | H3K79me2 per-bin share ratio distribution by eviction quartile |
inputs
[ "GSM8123438/439/440 NT", "GSM8123435/436/437 PCGF1-KO C1" ]
per arm — the numbers behind the chart
{
"K79_NT_VTP": {
"R_by_quartile": [
0.586,
0.8188,
0.928,
1.1865
],
"span_Q1_minus_Q4": -0.6005,
"median_per_bin_log2_ratio_by_quartile": [
-0.7213,
-0.168,
-0.0158,
0.2673
],
"median_per_bin_log2_spearman_vs_depth": -1,
"fraction_of_bins_below_1_by_quartile": [
0.8929,
0.6503,
0.5114,
0.2467
],
"n_bins_with_signal_in_both_tracks": [
308,
306,
307,
304
],
"largest_single_bin_share_of_quartile_DMSO": [
0.0066,
0.0084,
0.011,
0.0111
],
"bootstrap_R_ci95_by_quartile": [
[
0.5506,
0.6207
],
[
0.7789,
0.8567
],
[
0.885,
0.9734
],
[
1.145,
1.2272
]
],
"bootstrap_span_mean": -0.6001,
"bootstrap_span_ci95": [
-0.651,
-0.5446
],
"bootstrap_fraction_of_resamples_with_span_of_observed_sign": 1,
"leave_one_bin_out_worst_change_in_span": 0.0057
},
"K79_NT_SGC": {
"R_by_quartile": [
0.1396,
0.1453,
0.1452,
0.136
],
"span_Q1_minus_Q4": 0.0036,
"median_per_bin_log2_ratio_by_quartile": [
-2.8447,
-2.7107,
-2.6805,
-2.7389
],
"median_per_bin_log2_spearman_vs_depth": -0.4,
"fraction_of_bins_below_1_by_quartile": [
1,
0.9837,
0.9935,
0.9477
],
"n_bins_with_signal_in_both_tracks": [
308,
306,
307,
306
],
"largest_single_bin_share_of_quartile_DMSO": [
0.0066,
0.0084,
0.011,
0.0111
],
"bootstrap_R_ci95_by_quartile": [
[
0.1342,
0.1454
],
[
0.1391,
0.1517
],
[
0.1377,
0.1529
],
[
0.1267,
0.1461
]
],
"bootstrap_span_mean": 0.0034,
"bootstrap_span_ci95": [
-0.0078,
0.0138
],
"bootstrap_fraction_of_resamples_with_span_of_observed_sign": 0.7305,
"leave_one_bin_out_worst_change_in_span": 0.0014
},
"K79_C1_VTP": {
"R_by_quartile": [
0.6123,
0.7955,
0.8928,
1.0464
],
"span_Q1_minus_Q4": -0.4341,
"median_per_bin_log2_ratio_by_quartile": [
-0.633,
-0.2363,
-0.0837,
0.0713
],
"median_per_bin_log2_spearman_vs_depth": -1,
"fraction_of_bins_below_1_by_quartile": [
0.8669,
0.6984,
0.5733,
0.4169
],
"n_bins_with_signal_in_both_tracks": [
308,
305,
307,
307
],
"largest_single_bin_share_of_quartile_DMSO": [
0.0061,
0.008,
0.0093,
0.0099
],
"bootstrap_R_ci95_by_quartile": [
[
0.5759,
0.6474
],
[
0.7615,
0.8327
],
[
0.8556,
0.9295
],
[
1.0158,
1.0767
]
],
"bootstrap_span_mean": -0.4346,
"bootstrap_span_ci95": [
-0.4814,
-0.3897
],
"bootstrap_fraction_of_resamples_with_span_of_observed_sign": 1,
"leave_one_bin_out_worst_change_in_span": 0.0044
},
"K79_C1_SGC": {
"R_by_quartile": [
0.1334,
0.1325,
0.1349,
0.1225
],
"span_Q1_minus_Q4": 0.011,
"median_per_bin_log2_ratio_by_quartile": [
-2.8671,
-2.8406,
-2.8226,
-2.8885
],
"median_per_bin_log2_spearman_vs_depth": 0.2,
"fraction_of_bins_below_1_by_quartile": [
1,
0.9902,
0.9837,
0.9316
],
"n_bins_with_signal_in_both_tracks": [
308,
305,
307,
307
],
"largest_single_bin_share_of_quartile_DMSO": [
0.0061,
0.008,
0.0093,
0.0099
],
"bootstrap_R_ci95_by_quartile": [
[
0.1288,
0.1383
],
[
0.1272,
0.138
],
[
0.1281,
0.1423
],
[
0.1148,
0.1307
]
],
"bootstrap_span_mean": 0.0109,
"bootstrap_span_ci95": [
0.0018,
0.0203
],
"bootstrap_fraction_of_resamples_with_span_of_observed_sign": 0.991,
"leave_one_bin_out_worst_change_in_span": 0.0008
}
}- A bootstrap interval of width 0.106 on an unreplicated library reads as precision and is not. If this statistic is ever proposed as a pharmacodynamic endpoint, the interval that matters is the one across biological replicates, and this deposit cannot supply it.
- Do Q125's three marks survive the same per-bin battery? The code is written and the tracks are on disk in C:/Temp/q125; it was out of this beat's scope and is the cheapest remaining check in this whole line of work.
- Is there a correlated block of bins - one locus spanning many 2 kb bins - driving the Q1 loss? A per-chromosome or per-locus decomposition would answer it and was not run.
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.dataset
Q124's integrity criterion I0 is a whole-track check - its worst track value was 0.00013 of the non-blacklisted total - and so cannot detect a single bin dominating a 307-bin quartile's ratio.
Q124 re-analysis of GSE260742, committed cycle 83: criteria.I0_no_track_is_one_artefact_dominated: threshold 0.05, worst track TC_K79_VTP24 at 0.00013091, verdict PASS. analyses/q124-the-timecourse-and-the-pcgf1-knockout/results.json, read from the committed JSON rather than retyped. · Iqbal Lab oncofusion knowledge graph 2026What this evidence cannot show
Read from the committed JSON. I0 was correct for its own purpose and this is not a criticism of it; the scale of the check simply does not reach the scale of this beat's statistic.
Evidence that argues against this (1)
- A whole-track check and a within-quartile check could both pass and the result still be driven by a correlated block of bins - a single amplified locus spanning many 2 kb bins would defeat both. Iqbal Lab oncofusion knowledge graph 2026
- E2moderate confidence 3 of 5: One strong study, or several smaller consistent ones.dataset
For the control genotype under VTP-50469, the median per-bin log2 H3K79me2 share ratio across the four eviction quartiles is -0.7213 / -0.1680 / -0.0158 / +0.2673 (Spearman -1.000), the fraction of bins individually below 1 falls from 0.893 to 0.247, the largest single bin holds 0.66-1.11% of its quartile's DMSO signal, the 2,000-resample bin-level bootstrap 95% interval for the Q1-minus-Q4 span is [-0.651, -0.545] with 100% sign agreement, and leave-one-bin-out changes the span by at most 0.0057.
Q130 re-analysis of GSE260742: DEVIATION 1, deviation1-per-bin-robustness.json, results.K79_NT_VTP. analyses/q130-does-the-eviction-gradient-exist-in-h3k79me2/run.py, deviation1_per_bin_robustness.py, deviation2_two_criteria_i_wrote_badly.py, PREREGISTRATION.md and results.json, run 2026-09-23. Zero downloads: every track was binned by Q124 and read from disk. · Iqbal Lab oncofusion knowledge graph 2026What this evidence cannot show
NO SPIKE-IN NORMALISATION IS POSSIBLE. Q117 verified zero hits for the entire spike-in vocabulary across all 419,824 characters of GSE260742's 158 sample records and no scale factor is deposited, so every cross-condition number here is a WITHIN-SAMPLE SHARE. It cancels the unknown per-sample scale factor exactly and IS BLIND TO A UNIFORM CHANGE BY CONSTRUCTION: nothing here measures H3K79me2's abundance, everything measures its distribution. EXPOSURE ASYMMETRY: DMSO 72 h and SGC0946 5 uM 72 h are matched, VTP-50469 500 nM is 48 h, so every VTP ratio is a LOWER BOUND on the 72-hour effect - identical in both genotypes, so the genotype DIFFERENCE is unaffected. REPLICATION, CORRECTED MID-BEAT: the NTvKO arm deposits ONE H3K79me2 ChIP per genotype per drug, so ITS spans carry no technical error bar and its bin-level bootstrap intervals measure sampling within one library, NOT library-to-library variation. A REPLICATE PAIR DOES EXIST for a different culture - GSM8123372/374 and GSM8123386/388, both titled MLLAF9 Control, the second carrying `rep2` - already binned by Q122 and scored in Q130 DEVIATION 4: the SIGN and the monotonicity reproduce (Spearman -1.000 in all four murine VTP contrasts in this deposit) while the SPAN reproduces only to within a factor of 1.99 (-0.2438 against -0.4847). EVERY SPAN IN THIS BEAT SHOULD BE READ WITH THAT 2-FOLD REPRODUCIBILITY. THE KNOCKOUT is one CRISPR guide, one clone C1, no second clone, no rescue, and NO deposited PCGF1 protein or transcript measurement. S_FLAG is defined by a DYKDDDDK-tag ChIP on a RETROVIRALLY OVER-EXPRESSED tagged MLLAF9 construct, so it is the over-expressed molecule's sites and not an endogenous fusion's. THE H3K79me2 ANTIBODY IS NOT RESOLVED - `antibod` occurs zero times in the 158 GEO records - and is recorded unresolved rather than guessed. SYSTEM: murine MLL-AF9 (KMT2A::MLLT3) myeloid blood cells, NOT the infant B-lineage KMT2A::AFF1 disease this graph is about. POST HOC: none of these four statistics is in the pre-registration. A bin-level bootstrap measures bin sampling within ONE library and cannot speak to library-to-library variation, which is unmeasurable here.
Evidence that argues against this (2)
- A published methods study establishes that STANDARD ChIP-seq normalisation FAILS TO DETECT a genome-wide decrease in a histone mark under an inhibitor of its writer: standard methods see no H3K27me3 reduction under an EZH2 inhibitor, and a substantial reduction appears only with a Drosophila chromatin spike-in. Every number in this entry is exactly the kind of normalisation that paper shows to be blind. Egan B et al. 2016
- The system is murine MLL-AF9 (KMT2A::MLLT3) myeloid cells carrying a retrovirally over-expressed FLAG-tagged construct. This graph exists for human infant B-lineage KMT2A::AFF1, and this corpus's commonest documented synthesis failure is generalising a murine MLL-AF9 myeloid result to that disease. Neville D et al. 2026
- E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.dataset
The same battery applied to the DOT1L-inhibitor arm returns a negative: under SGC0946 the median per-bin log2 ratio is -2.8447 / -2.7107 / -2.6805 / -2.7389 with Spearman -0.400, the bootstrap span interval is [-0.0078, +0.0138] spanning zero, and only 73.1% of 2,000 resamples keep the observed sign.
Q130 re-analysis of GSE260742: DEVIATION 1, results.K79_NT_SGC. analyses/q130-does-the-eviction-gradient-exist-in-h3k79me2/run.py, deviation1_per_bin_robustness.py, deviation2_two_criteria_i_wrote_badly.py, PREREGISTRATION.md and results.json, run 2026-09-23. Zero downloads: every track was binned by Q124 and read from disk. · Iqbal Lab oncofusion knowledge graph 2026What this evidence cannot show
A battery that returns a negative on one arm and a positive on another in the same bins is behaving as an instrument should, but the two arms differ in exposure (72 h against 48 h) as well as in drug, so this is not a perfectly controlled contrast.
Evidence that argues against this (1)
- Failing to detect a gradient under SGC0946 is not proof of its absence, and the design's power against a small gradient is not quantified. Iqbal Lab oncofusion knowledge graph 2026
the per-bin robustness of the headline gradient
the same battery returns a negative on the DOT1L arm