表达矩阵处理—表达QC(reads)

2020-03-31 12:30:37 浏览数 (1)
7. 清理表达矩阵

7.2

表达QC（reads）

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library(SingleCellExperiment)
library(scater)
options(stringsAsFactors = FALSE)
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reads <- read.table("tung/reads.txt", sep = "t")
anno <- read.table("tung/annotation.txt", sep = "t", header = TRUE)
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head(reads[ , 1:3])
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##                 NA19098.r1.A01 NA19098.r1.A02 NA19098.r1.A03
## ENSG00000237683              0              0              0
## ENSG00000187634              0              0              0
## ENSG00000188976             57            140              1
## ENSG00000187961              0              0              0
## ENSG00000187583              0              0              0
## ENSG00000187642              0              0              0
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head(anno)
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##   individual replicate well      batch      sample_id
## 1    NA19098        r1  A01 NA19098.r1 NA19098.r1.A01
## 2    NA19098        r1  A02 NA19098.r1 NA19098.r1.A02
## 3    NA19098        r1  A03 NA19098.r1 NA19098.r1.A03
## 4    NA19098        r1  A04 NA19098.r1 NA19098.r1.A04
## 5    NA19098        r1  A05 NA19098.r1 NA19098.r1.A05
## 6    NA19098        r1  A06 NA19098.r1 NA19098.r1.A06
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reads <- SingleCellExperiment(
    assays = list(counts = as.matrix(reads)),
    colData = anno
)
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keep_feature <- rowSums(counts(reads) > 0) > 0
reads <- reads[keep_feature, ]
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isSpike(reads, "ERCC") <- grepl("^ERCC-", rownames(reads))
isSpike(reads, "MT") <- rownames(reads) %in%
    c("ENSG00000198899", "ENSG00000198727", "ENSG00000198888",
    "ENSG00000198886", "ENSG00000212907", "ENSG00000198786",
    "ENSG00000198695", "ENSG00000198712", "ENSG00000198804",
    "ENSG00000198763", "ENSG00000228253", "ENSG00000198938",
    "ENSG00000198840")
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reads <- calculateQCMetrics(
    reads,
    feature_controls = list(
        ERCC = isSpike(reads, "ERCC"),
        MT = isSpike(reads, "MT")
    )
)
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hist(
    reads$total_counts,
    breaks = 100
)
abline(v = 1.3e6, col = "red")
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filter_by_total_counts <- (reads$total_counts > 1.3e6)
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table(filter_by_total_counts)
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## filter_by_total_counts
## FALSE  TRUE
##   180   684
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hist(
    reads$total_features,
    breaks = 100
)
abline(v = 7000, col = "red")
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filter_by_expr_features <- (reads$total_features > 7000)
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table(filter_by_expr_features)
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## filter_by_expr_features
## FALSE  TRUE
##   116   748
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plotPhenoData(
    reads,
    aes_string(
        x = "total_features",
        y = "pct_counts_MT",
        colour = "batch"
    )
)
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plotPhenoData(
    reads,
    aes_string(
        x = "total_features",
        y = "pct_counts_ERCC",
        colour = "batch"
    )
)
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filter_by_ERCC <-
    reads$batch != "NA19098.r2" & reads$pct_counts_ERCC < 25
table(filter_by_ERCC)
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## filter_by_ERCC
## FALSE  TRUE
##   103   761
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filter_by_MT <- reads$pct_counts_MT < 30
table(filter_by_MT)
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## filter_by_MT
## FALSE  TRUE
##    18   846
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reads$use <- (
    # sufficient features (genes)
    filter_by_expr_features &
    # sufficient molecules counted
    filter_by_total_counts &
    # sufficient endogenous RNA
    filter_by_ERCC &
    # remove cells with unusual number of reads in MT genes
    filter_by_MT
)
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table(reads$use)
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##
## FALSE  TRUE
##   258   606
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reads <- plotPCA(
    reads,
    size_by = "total_features",
    shape_by = "use",
    pca_data_input = "pdata",
    detect_outliers = TRUE,
    return_SCE = TRUE
)
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table(reads$outlier)
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##
## FALSE  TRUE
##   756   108
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library(limma)
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##
## Attaching package: 'limma'
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## The following object is masked from 'package:scater':
##
##     plotMDS
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## The following object is masked from 'package:BiocGenerics':
##
##     plotMA
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auto <- colnames(reads)[reads$outlier]
man <- colnames(reads)[!reads$use]
venn.diag <- vennCounts(
    cbind(colnames(reads) %in% auto,
    colnames(reads) %in% man)
)
vennDiagram(
    venn.diag,
    names = c("Automatic", "Manual"),
    circle.col = c("blue", "green")
)
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plotQC(reads, type = "highest-expression")
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filter_genes <- apply(
    counts(reads[, colData(reads)$use]),
    1,
    function(x) length(x[x > 1]) >= 2
)
rowData(reads)$use <- filter_genes
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table(filter_genes)
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## filter_genes
## FALSE  TRUE
##  2664 16062
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dim(reads[rowData(reads)$use, colData(reads)$use])
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## [1] 16062   606
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assay(reads, "logcounts_raw") <- log2(counts(reads)   1)
reducedDim(reads) <- NULL
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saveRDS(reads, file = "tung/reads.rds")

通过比较图7.6和图7.13，很明显基于read的过滤比基于UMI的分析去除了更多的细胞。如果您返回并比较结果，您应该能够得出结论，ERCC和MT过滤器对于基于read的分析更严格。

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sessionInfo()
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## R version 3.4.3 (2017-11-30)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Debian GNU/Linux 9 (stretch)
##
## Matrix products: default
## BLAS: /usr/lib/openblas-base/libblas.so.3
## LAPACK: /usr/lib/libopenblasp-r0.2.19.so
##
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=C
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] parallel  stats4    methods   stats     graphics  grDevices utils
## [8] datasets  base
##
## other attached packages:
##  [1] limma_3.34.9               scater_1.6.3
##  [3] ggplot2_2.2.1              SingleCellExperiment_1.0.0
##  [5] SummarizedExperiment_1.8.1 DelayedArray_0.4.1
##  [7] matrixStats_0.53.1         Biobase_2.38.0
##  [9] GenomicRanges_1.30.3       GenomeInfoDb_1.14.0
## [11] IRanges_2.12.0             S4Vectors_0.16.0
## [13] BiocGenerics_0.24.0        knitr_1.20
##
## loaded via a namespace (and not attached):
##   [1] backports_1.1.2        plyr_1.8.4             lazyeval_0.2.1
##   [4] sp_1.2-7               shinydashboard_0.6.1   splines_3.4.3
##   [7] digest_0.6.15          htmltools_0.3.6        viridis_0.5.0
##  [10] magrittr_1.5           memoise_1.1.0          cluster_2.0.6
##  [13] prettyunits_1.0.2      colorspace_1.3-2       blob_1.1.0
##  [16] rrcov_1.4-3            xfun_0.1               dplyr_0.7.4
##  [19] RCurl_1.95-4.10        tximport_1.6.0         lme4_1.1-15
##  [22] bindr_0.1              zoo_1.8-1              glue_1.2.0
##  [25] gtable_0.2.0           zlibbioc_1.24.0        XVector_0.18.0
##  [28] MatrixModels_0.4-1     car_2.1-6              kernlab_0.9-25
##  [31] prabclus_2.2-6         DEoptimR_1.0-8         SparseM_1.77
##  [34] VIM_4.7.0              scales_0.5.0           sgeostat_1.0-27
##  [37] mvtnorm_1.0-7          DBI_0.7                GGally_1.3.2
##  [40] edgeR_3.20.9           Rcpp_0.12.15           sROC_0.1-2
##  [43] viridisLite_0.3.0      xtable_1.8-2           progress_1.1.2
##  [46] laeken_0.4.6           bit_1.1-12             mclust_5.4
##  [49] vcd_1.4-4              httr_1.3.1             RColorBrewer_1.1-2
##  [52] fpc_2.1-11             modeltools_0.2-21      pkgconfig_2.0.1
##  [55] reshape_0.8.7          XML_3.98-1.10          flexmix_2.3-14
##  [58] nnet_7.3-12            locfit_1.5-9.1         labeling_0.3
##  [61] rlang_0.2.0            reshape2_1.4.3         AnnotationDbi_1.40.0
##  [64] munsell_0.4.3          tools_3.4.3            RSQLite_2.0
##  [67] pls_2.6-0              evaluate_0.10.1        stringr_1.3.0
##  [70] cvTools_0.3.2          yaml_2.1.17            bit64_0.9-7
##  [73] robustbase_0.92-8      bindrcpp_0.2           nlme_3.1-129
##  [76] mime_0.5               quantreg_5.35          biomaRt_2.34.2
##  [79] compiler_3.4.3         pbkrtest_0.4-7         beeswarm_0.2.3
##  [82] e1071_1.6-8            tibble_1.4.2           robCompositions_2.0.6
##  [85] pcaPP_1.9-73           stringi_1.1.6          highr_0.6
##  [88] lattice_0.20-34        trimcluster_0.1-2      Matrix_1.2-7.1
##  [91] nloptr_1.0.4           pillar_1.2.1           lmtest_0.9-35
##  [94] data.table_1.10.4-3    cowplot_0.9.2          bitops_1.0-6
##  [97] httpuv_1.3.6.1         R6_2.2.2               bookdown_0.7
## [100] gridExtra_2.3          vipor_0.4.5            boot_1.3-18
## [103] MASS_7.3-45            assertthat_0.2.0       rhdf5_2.22.0
## [106] rprojroot_1.3-2        rjson_0.2.15           GenomeInfoDbData_1.0.0
## [109] diptest_0.75-7         mgcv_1.8-23            grid_3.4.3
## [112] class_7.3-14           minqa_1.2.4            rmarkdown_1.8
## [115] mvoutlier_2.0.9        shiny_1.0.5            ggbeeswarm_0.6.0
umi

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