# Fast MaxLFQ

#### Thang V Pham

#### 2024-12-03

We have implemented a highly optimized version of the iq pipeline (Pham et al., Bioinformatics 2020). To run the following examples, download DIA-report-long-format.zip and unzip the file to a local working directory.

The unzipped file `DIA-report-long-format.txt` is a tab-deliminated text file exported from a Spectronaut search using this export schema iq.rs. The user might want to import this schema to their Spectronaut installation for the ease of using the iq pipeline.

## The standard pipeline

First, we apply the standard pipeline implemented in pure R. Read and filter the data

    library("iq") # if not already installed, run install.packages("iq")

    raw <- read.delim("DIA-Report-long-format.txt")

    selected <- raw$F.ExcludedFromQuantification == "False" &
                !is.na(raw$PG.Qvalue) & (raw$PG.Qvalue < 0.01) &
                !is.na(raw$EG.Qvalue) & (raw$EG.Qvalue < 0.01)

    raw <- raw[selected,]

Normalize the data, create a protein list, and perform the MaxLFQ algorithm

    sample_id  <- "R.FileName"

    secondary_id <- c("EG.Library", "FG.Id", "FG.Charge", "F.FrgIon", "F.Charge", "F.FrgLossType")

    norm_data <- iq::preprocess(raw,
                                sample_id  = sample_id,
                                secondary_id = secondary_id)
    #> Concatenating secondary ids...
    #> Removing low intensities...
    #> Barplotting raw data ...
    #> Median normalization ...
    #> Barplotting after normalization ...

    protein_list <- iq::create_protein_list(norm_data)
    #> # proteins = 3554, # samples = 24
    #> 5%
    #> 10%
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    #> 80%
    #> 85%
    #> 90%
    #> 95%
    #> 100%
    #> Completed.

    result <- iq::create_protein_table(protein_list)
    #> 5%
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    #> 25%
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    #> Completed.

Extract annotation columns and write the result to an output file

    annotation_columns <- c("PG.Genes", "PG.ProteinNames")

    extra_names <- iq::extract_annotation(rownames(result$estimate),
                                          raw,
                                          annotation_columns = annotation_columns)

    write.table(cbind(Protein = rownames(result$estimate),
                      extra_names[, annotation_columns],
                      MaxLFQ_annotation = result$annotation,
                      result$estimate),
                "iq-MaxLFQ.txt", sep = "\t", row.names = FALSE)

The resulting file `iq-MaxLFQ.txt` is the protein level quantification report in a tab-deliminated text format.

## A faster MaxLFQ implementation

The function `iq::fast_MaxLFQ` implemented in C++ combines the functionalities of `iq::create_protein_list` and `iq::create_protein_table`.

    #--------------------- Replacing ---------------------
    # protein_list <- iq::create_protein_list(norm_data) #
    # result <- iq::create_protein_table(protein_list)   #
    #-----------------------------------------------------

    result_faster <- iq::fast_MaxLFQ(norm_data)
    #> nrow = 3369557, # proteins = 3554, # samples = 24
    #> Using 35 threads...
    #> 0%
    #> 6%
    #> 13%
    #> 18%
    #> 24%
    #> 29%
    #> 36%
    #> 42%
    #> 47%
    #> 52%
    #> 57%
    #> 64%
    #> 70%
    #> 77%
    #> 82%
    #> 87%
    #> 92%
    #> 98%
    #> Completed.

The results of the R implementation `result` and C++ implementation `result_faster` should be equal up to the floating-point precision of the underlying numerical libraries.

    cat("Max difference =", max(abs(result_faster$estimate - result$estimate), na.rm = TRUE), "\n")
    #> Max difference = 1.332268e-13

    cat("Identical NAs =", identical(is.na(result_faster$estimate), is.na(result$estimate)), "\n")
    #> Identical NAs = TRUE

    cat("Equal annotation =", identical(result_faster$annotation, result$annotation), "\n")
    #> Equal annotation = TRUE

### Benchmarking execution time

We can check the improvement in execution time. The following result is obtained on a computer with 12 CPU cores.

    system.time({
        protein_list <- iq::create_protein_list(norm_data)
        result <- iq::create_protein_table(protein_list)
    })
    #> # proteins = 3554, # samples = 24
    #> 5%
    #> 10%
    #> 15%
    #> 20%
    #> 25%
    #> 30%
    #> 35%
    #> 40%
    #> 45%
    #> 50%
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    #> 60%
    #> 65%
    #> 70%
    #> 75%
    #> 80%
    #> 85%
    #> 90%
    #> 95%
    #> 100%
    #> Completed.
    #> 5%
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    #> 75%
    #> 80%
    #> 85%
    #> 90%
    #> 95%
    #> 100%
    #> Completed.
    #>    user  system elapsed
    #>  395.82    9.00  404.87

    system.time({
        result_faster <- iq::fast_MaxLFQ(norm_data)
    })
    #> nrow = 3369557, # proteins = 3554, # samples = 24
    #> Using 35 threads...
    #> 0%
    #> 7%
    #> 15%
    #> 24%
    #> 30%
    #> 36%
    #> 42%
    #> 47%
    #> 54%
    #> 59%
    #> 65%
    #> 72%
    #> 77%
    #> 83%
    #> 90%
    #> 96%
    #> Completed.
    #>    user  system elapsed
    #>    7.75    0.04    3.55

## An efficient data structure and data loading

We have implemented a fast data loading algorithm and an efficient data structure. The memory usage is highly optimized to enable the processing of very large datasets.

    sample_id  <- "R.FileName"

    secondary_id <- c("EG.Library", "FG.Id", "FG.Charge", "F.FrgIon", "F.Charge", "F.FrgLossType")

    annotation_columns <- c("PG.Genes", "PG.ProteinNames")

    iq_dat <- iq::fast_read("DIA-report-long-format.txt",
                            sample_id  = sample_id,
                            secondary_id = secondary_id,
                            filter_string_equal = c("F.ExcludedFromQuantification" = "False"),
                            annotation_col = annotation_columns)
    #>
    #> Command: --sample R.FileName --primary PG.ProteinGroups --secondary EG.Library FG.Id FG.Charge F.FrgIon F.Charge F.FrgLossType --quant F.PeakArea --annotation PG.Genes PG.ProteinNames --filter-string-equal F.ExcludedFromQuantification False --filter-double-less PG.Qvalue 0.01 --filter-double-less EG.Qvalue 0.01 DIA-report-long-format.txt
    #>
    #> Sample column:
    #>     R.FileName
    #> Protein column:
    #>     PG.ProteinGroups
    #> Ion column(s):
    #>     EG.Library FG.Id FG.Charge F.FrgIon F.Charge F.FrgLossType
    #> Quant column:
    #>     F.PeakArea
    #> Annotation column(s):
    #>     PG.Genes PG.ProteinNames
    #> String equal filter(s):
    #>     F.ExcludedFromQuantification == False
    #> Double less filter(s):
    #>     PG.Qvalue < 0.010000
    #>     EG.Qvalue < 0.010000
    #>
    #> Using 4 threads ...
    #> 20 samples read
    #>
    #> # lines read (excluding headers)      = 5547331
    #> # quantitative values after filtering = 3390569
    #>
    #> # samples  = 24
    #> # proteins = 3554

    iq_norm_data <- iq::fast_preprocess(iq_dat$quant_table)
    #> Removing low intensities...
    #> Barplotting raw data ...
    #> Median normalization ...
    #> Barplotting after normalization ...

    result_fastest <- iq::fast_MaxLFQ(iq_norm_data,
                                      row_names = iq_dat$protein[, 1],
                                      col_names = iq_dat$sample)
    #> nrow = 3369557, # proteins = 3554, # samples = 24
    #> Using 35 threads...
    #> 0%
    #> 5%
    #> 11%
    #> 16%
    #> 22%
    #> 27%
    #> 33%
    #> 40%
    #> 46%
    #> 53%
    #> 58%
    #> 63%
    #> 69%
    #> 87%
    #> 93%
    #> 99%
    #> Completed.

The result of the optimized pipeline `result_fastest` should be the same as that of the standard pipeline `result`.

    cat("Max difference =", max(abs(result_fastest$estimate - result$estimate), na.rm = TRUE), "\n")
    #> Max difference = 1.136868e-13

    cat("Identical NAs =", identical(is.na(result_fastest$estimate), is.na(result$estimate)), "\n")
    #> Identical NAs = TRUE

    cat("Equal annotation =", identical(result_fastest$annotation, result$annotation), "\n")
    #> Equal annotation = TRUE

The annotation columns are stored in the `protein` component of the input data structure `iq_dat`. We can extract the annotation columns and write the result to an output text file.

    iq_extra_names <- iq::extract_annotation(rownames(result_fastest$estimate),
                                             iq_dat$protein,
                                             annotation_columns = annotation_columns)

    write.table(cbind(Protein = rownames(result_fastest$estimate),
                      iq_extra_names[, annotation_columns],
                      MaxLFQ_annotation = result_fastest$annotation,
                      result_fastest$estimate),
                "iq-MaxLFQ-fast.txt", sep = "\t", row.names = FALSE)

### Benchmarking execution time

    sample_id  <- "R.FileName"

    secondary_id <- c("EG.Library", "FG.Id", "FG.Charge", "F.FrgIon", "F.Charge", "F.FrgLossType")

    annotation_columns <- c("PG.Genes", "PG.ProteinNames")

    system.time({

        # reading data
        raw <- read.delim("DIA-report-long-format.txt")

        # filtering
        selected <- raw$F.ExcludedFromQuantification == "False" &
                    !is.na(raw$PG.Qvalue) & raw$PG.Qvalue < 0.01 &
                    !is.na(raw$EG.Qvalue) & raw$EG.Qvalue < 0.01

        raw <- raw[selected,]

        ## process

        norm_data <- iq::preprocess(raw,
                                    sample_id  = sample_id,
                                    secondary_id = secondary_id)

        protein_list <- iq::create_protein_list(norm_data)

        result <- iq::create_protein_table(protein_list)

    })
    #> Concatenating secondary ids...
    #> Removing low intensities...
    #> Barplotting raw data ...
    #> Median normalization ...
    #> Barplotting after normalization ...
    #> # proteins = 3554, # samples = 24
    #> 5%
    #> 10%
    #> 15%
    #> 20%
    #> 25%
    #> 30%
    #> 35%
    #> 40%
    #> 45%
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    #> 60%
    #> 65%
    #> 70%
    #> 75%
    #> 80%
    #> 85%
    #> 90%
    #> 95%
    #> 100%
    #> Completed.
    #> 5%
    #> 10%
    #> 15%
    #> 20%
    #> 25%
    #> 30%
    #> 35%
    #> 40%
    #> 45%
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    #> 60%
    #> 65%
    #> 70%
    #> 75%
    #> 80%
    #> 85%
    #> 90%
    #> 95%
    #> 100%
    #> Completed.
    #>    user  system elapsed
    #>  569.83   14.78  584.75

    system.time({
        iq_dat <- iq::fast_read("DIA-report-long-format.txt",
                                sample_id  = sample_id,
                                secondary_id = secondary_id,
                                filter_string_equal = c("F.ExcludedFromQuantification" = "False"),
                                annotation_col = annotation_columns)

        iq_norm_data <- iq::fast_preprocess(iq_dat$quant_table)

        result_fastest <- iq::fast_MaxLFQ(iq_norm_data,
                                          row_names = iq_dat$protein[, 1],
                                          col_names = iq_dat$sample)
    })
    #>
    #> Command: --sample R.FileName --primary PG.ProteinGroups --secondary EG.Library FG.Id FG.Charge F.FrgIon F.Charge F.FrgLossType --quant F.PeakArea --annotation PG.Genes PG.ProteinNames --filter-string-equal F.ExcludedFromQuantification False --filter-double-less PG.Qvalue 0.01 --filter-double-less EG.Qvalue 0.01 DIA-report-long-format.txt
    #>
    #> Sample column:
    #>     R.FileName
    #> Protein column:
    #>     PG.ProteinGroups
    #> Ion column(s):
    #>     EG.Library FG.Id FG.Charge F.FrgIon F.Charge F.FrgLossType
    #> Quant column:
    #>     F.PeakArea
    #> Annotation column(s):
    #>     PG.Genes PG.ProteinNames
    #> String equal filter(s):
    #>     F.ExcludedFromQuantification == False
    #> Double less filter(s):
    #>     PG.Qvalue < 0.010000
    #>     EG.Qvalue < 0.010000
    #>
    #> Using 4 threads ...
    #> 20 samples read
    #>
    #> # lines read (excluding headers)      = 5547331
    #> # quantitative values after filtering = 3390569
    #>
    #> # samples  = 24
    #> # proteins = 3554
    #> Removing low intensities...
    #> Barplotting raw data ...
    #> Median normalization ...
    #> Barplotting after normalization ...
    #> nrow = 3369557, # proteins = 3554, # samples = 24
    #> Using 35 threads...
    #> 0%
    #> 5%
    #> 13%
    #> 18%
    #> 24%
    #> 29%
    #> 34%
    #> 40%
    #> 45%
    #> 50%
    #> 55%
    #> 61%
    #> 68%
    #> 75%
    #> 80%
    #> 86%
    #> 92%
    #> 98%
    #> Completed.
    #>    user  system elapsed
    #>   27.54    1.56   12.34

## References

1. Pham TV, Henneman AA, Jimenez CR (2020) iq: an R package to estimate relative protein abundances from ion quantification in DIA-MS-based proteomics. Bioinformatics 36(8):2611-2613.