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Update readme with newer results, new menu options

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eugenefischer
2022-10-01 13:00:33 -05:00
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@@ -128,7 +128,8 @@ By default, the Options menu looks like this:
3) Turn on graph/data file caching
4) Turn off serialized binary graph output
5) Turn on GraphML graph output
6) Maximum weight matching algorithm options
6) Turn on calculation of p-values
7) Maximum weight matching algorithm options
0) Return to main menu
```
@@ -182,7 +183,7 @@ Structure:
| Alpha CDR3 | Beta CDR3 | Alpha CDR1 | Beta CDR1 |
|---|---|---|---|
|unique number|unique number|number|number|
| ... | ... |... | ... |
---
#### Sample Plate Files
@@ -191,7 +192,8 @@ described above). The wells are filled randomly from a Cell Sample file, accordi
frequency distribution. Additionally, every individual sequence within each cell may, with some
given dropout probability, be omitted from the file; this simulates the effect of amplification errors
prior to sequencing. Plates can also be partitioned into any number of sections, each of which can have a
different concentration of T cells per well.
different concentration of T cells per well. Alternatively, the number of T cells in each well can be randomized between
given minimum and maximum population values.
Options when making a Sample Plate file:
* Cell Sample file to use
@@ -201,7 +203,7 @@ Options when making a Sample Plate file:
* Standard deviation size
* Exponential
* Lambda value
* *(Based on the slope of the graph in Figure 4C of the pairSEQ paper, the distribution of the original experiment was approximately exponential with a lambda ~0.6. (Howie, et al. 2015))*
* *(Based on the slope of the graph in Figure 4C of the pairSEQ paper, the distribution of the original experiment was very roughly exponential with a lambda ~0.6. (Howie, et al. 2015) The actual distribution was certainly quite different.)*
* Total number of wells on the plate
* Well populations random or fixed
* If random, minimum and maximum population sizes
@@ -248,12 +250,12 @@ then use it for multiple different BiGpairSEQ simulations.
Options for creating a Graph/Data file:
* The Cell Sample file to use
* The Sample Plate file to use. (This must have been generated from the selected Cell Sample file.)
* Whether to simulate sequence read depth. If simulated:
* The read depth (number of times each sequence is read)
* The read error rate (probability a sequence is misread)
* The error collision rate (probability two misreads produce the same spurious sequence)
* The real sequence collision rate (probability that a misread will produce a different, real sequence from the sample plate. Only applies to new misreads; once an error of this type has occurred, it's likelihood of ocurring again is dominated by the error collision probability.)
* The Sample Plate file to use (This must have been generated from the selected Cell Sample file.)
* Whether to simulate sequencing read depth. If simulated:
* The read depth (The number of times each sequence is read)
* The read error rate (The probability a sequence is misread)
* The error collision rate (The probability two misreads produce the same spurious sequence)
* The real sequence collision rate (The probability that a misread will produce a different, real sequence from the sample plate. Only applies to new misreads; once an error of this type has occurred, it's likelihood of occurring again is dominated by the error collision probability.)
These files do not have a human-readable structure, and are not portable to other programs.
@@ -270,7 +272,7 @@ compare the matching results to the original Cell Sample .csv file.
#### Matching Results Files
Matching results files consist of the results of a BiGpairSEQ matching simulation. Making them requires a serialized
binary Graph/Data file (.ser). (Because .graphML files are larger than .ser files, BiGpairSEQ_Sim supports .graphML
output only. Graph/data input must use a serialized binary.)
output only. Graph input must use a serialized binary.)
Matching results files are in CSV format. Rows are sequence pairings with extra relevant data. Columns are pairing-specific details.
Metadata about the matching simulation is included as comments. Comments are preceded by `#`.
@@ -288,56 +290,83 @@ Options when running a BiGpairSEQ simulation of CDR3 alpha/beta matching:
Example output:
```
# Source Sample Plate file: 4MilCellsPlate.csv
# Source Graph and Data file: 4MilCellsPlateGraph.ser
# T cell counts in sample plate wells: 30000
# Total alphas found: 11813
# Total betas found: 11808
# High overlap threshold: 94
# Low overlap threshold: 3
# Minimum overlap percent: 0
# Maximum occupancy difference: 96
# Pairing attempt rate: 0.438
# Correct pairings: 5151
# Incorrect pairings: 18
# Pairing error rate: 0.00348
# Simulation time: 862 seconds
# sample plate filename: 8MilCells_Plate.csv
# sequence dropout rate: 0.1
# graph filename: 8MilGraph_rd2
# MWM algorithm type: LEDA book with heap: FIBONACCI
# matching weight: 218017.0
# well populations: 4000
# sequence read depth: 100
# sequence read error rate: 0.01
# read error collision rate: 0.1
# real sequence collision rate: 0.05
# total alphas read from plate: 323711
# total betas read from plate: 323981
# pre-filter sequences present in all wells: true
# pre-filter sequences based on occupancy/read count discrepancy: true
# alphas in graph (after pre-filtering): 11707
# betas in graph (after pre-filtering): 11705
# high overlap threshold for pairing: 95
# low overlap threshold for pairing: 3
# minimum overlap percent for pairing: 0
# maximum occupancy difference for pairing: 2147483647
# pairing attempt rate: 0.716
# correct pairing count: 8373
# incorrect pairing count: 7
# pairing error rate: 0.000835
# time to generate graph (seconds): 293
# time to pair sequences (seconds): 1,416
# total simulation time (seconds): 1,709
```
| Alpha | Alpha well count | Beta | Beta well count | Overlap count | Matched Correctly? | P-value |
|---|---|---|---|---|---|---|
|5242972|17|1571520|18|17|true|1.41E-18|
|5161027|18|2072219|18|18|true|7.31E-20|
|4145198|33|1064455|30|29|true|2.65E-21|
|7700582|18|112748|18|18|true|7.31E-20|
|10258642|73|1172093|72|70.0|true|4.19E-21|
|6186865|34|4290363|37|34.0|true|4.56E-26|
|10222686|70|11044018|72|68.0|true|9.55E-25|
|5338100|75|2422988|76|74.0|true|4.57E-22|
|12363907|33|6569852|35|33.0|true|5.70E-26|
|...|...|...|...|...|...|...|
---
**NOTE: The p-values in the output are not used for matching**—they aren't part of the BiGpairSEQ algorithm at all.
P-values are calculated *after* BiGpairSEQ matching is completed, for purposes of comparison only,
using the (2021 corrected) formula from the original pairSEQ paper. (Howie, et al. 2015)
**NOTE: The p-values in the sample output abpve are not used for matching**—they aren't part of the BiGpairSEQ algorithm at all.
P-values (if enabled in the interactive menu options or by using the -pv flag in the command line) are calculated *after*
BiGpairSEQ matching is completed, for purposes of comparison with pairSEQ only, using the (corrected) formula from the
original pairSEQ paper. (Howie, et al. 2015) Calculation of p-values is off by default to reduce processing time.
## PERFORMANCE (old results; need updating to reflect current, improved simulator performance)
## PERFORMANCE
On a home computer with a Ryzen 5600X CPU, 64GB of 3200MHz DDR4 RAM (half of which was allocated to the Java Virtual Machine), and a PCIe 3.0 SSD, running Linux Mint 20.3 Edge (5.13 kernel),
the author ran a BiGpairSEQ simulation of a 96-well sample plate with 30,000 T cells/well comprising ~11,800 alphas and betas,
taken from a sample of 4,000,000 distinct cells with an exponential frequency distribution (lambda 0.6).
Several BiGpairSEQ simulations were performed on a home computer with the following specs:
With min/max occupancy threshold of 3 and 94 wells for matching, and no other pre-filtering, BiGpairSEQ identified 5,151
correct pairings and 18 incorrect pairings, for an accuracy of 99.652%.
* Ryzen 5600X CPU
* 128GB of 3200MHz DDR4 RAM
* 2TB PCIe 3.0 SSD
* Linux Mint 21 (5.15 kernel)
The total simulation time was 14'22". If intermediate results were held in memory, this would be equivalent to the total elapsed time.
### Simulation 1
This simulation was an attempt to replicate the conditions of experiment 1 from the 2015 pairSEQ paper: a matching was found for a
96-well sample plate with 4,000 T cells/well comprising ~11,900 TCRAs and TCRBs, taken from a sample of 8,400,000
distinct cells with an exponential frequency distribution (lambda 0.6). The sequence dropout rate was 10%, as the analysis
from the original paper concluded that most TCR sequences "have less than a 10% chance of going unobserved." (Howie, et al. 2015)
Since this implementation of BiGpairSEQ writes intermediate results to disk (to improve the efficiency of *repeated* simulations
with different filtering options), the actual elapsed time was greater. File I/O time was not measured, but took
slightly less time than the simulation itself. Real elapsed time from start to finish was under 30 minutes.
The original paper does not contain (or the author of this document failed to identify) information on sequencing depth,
read error probability, or the probabilities of different kinds of read error collisions. As the pre-filtering of BiGpairSEQ
has successfully filtered out all such errors for any reasonable error rates the author has yet tested, this simulation was
done without any sequencing errors, to reduce the processing time.
As mentioned in the theory section, performance could be improved by implementing a more efficient algorithm for finding
the maximum weight matching.
With min/max occupancy thresholds of 3 and 95 wells respectively for matching, BiGpairSEQ identified:
* 8,495 correct pairings
* 5 incorrect pairings
## BEHAVIOR WITH RANDOMIZED WELL POPULATIONS
for an overall pairing accuracy of 99.9992%.
The total simulation time (excluding file I/O) was 28m52. The total elapsed time with file I/O was 41m23s.
Calculation of p-values was enabled for this simulation, increasing the overall processing time.
## BEHAVIOR WITH RANDOMIZED WELL POPULATIONS (old results, need updating for new version of the simulator (though resilience to varying well populations is unchanged))
A series of BiGpairSEQ simulations were conducted using a cell sample file of 3.5 million unique T cells. From these cells,
10 sample plate files were created. All of these sample plates had 96 wells, used an exponential distribution with a lambda of 0.6, and