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Question: dba.count generates less ranges than input peakset
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gravatar for pau.rodriguez.sodupe
7 months ago by
pau.rodriguez.sodupe0 wrote:

Hi,

I've been analyzing ATAC-seq data with DiffBind. I have generated a dba object with the samples' bams and the peak bed files resulting from macs. I would like to take advantage of this dba object in order to get the normalized counts of my samples for a pre-defined peakset I have builded myself (custom_peakset). I don't know why when I retrieve the peakset, after computing the count using custom_peakset, it returns 94 ranges since my peakset is a GRanges Object with 288 ranges. I was wondering if the ranges without reads in non of my samples where omitted, but looking for couple of examples I have realized that those omitted ranges have coverage in my samples. What's wrong with my approach?

 

dba object (WT_KO_data)

5 Samples, 71116 sites in matrix (96868 total):

     ID Tissue Factor Condition Treatment Replicate Caller Intervals

1 CWS37  Mouse   CD34        WT       AUL         1    bed     61132

2 CWS38  Mouse   CD34        WT       AUL         2    bed     75585

3 CWS41  Mouse   CD34        WT       AUL         3    bed     56091

4 CSJ87  Mouse   CD34        KO       AUL         1    bed     65864

5 CSJ89  Mouse   CD34        KO       AUL         2    bed     65802

custom_peakset (GRanges_TSS_DE_genes)

GRanges object with 288 ranges and 1 metadata column:

        seqnames                 ranges strand |    name2

           <Rle>              <IRanges>  <Rle> | <factor>

    139     chr1 [186621791, 186623791]      * |    Tgfb2

    266     chr1 [ 50926522,  50928522]      * |   Tmeff2

    352     chr1 [ 73909230,  73911230]      * |     Tns1

    375     chr1 [ 83254443,  83256443]      * |   Sphkap

    409     chr1 [ 90765859,  90767859]      * |   Col6a3

    ...      ...                    ...    ... .      ...

  40470    chr19   [27215483, 27217483]      * |    Vldlr

  40523    chr19   [34239335, 34241335]      * |    Acta2

  40648    chr19   [44393449, 44395449]      * |     Scd1

  40723    chr19   [46598105, 46600105]      * |    Wbp1l

  40748    chr19   [47730755, 47732755]      * |     Sfr1

  -------

code:

WT_KO_TSS_counts <- dba.count(WT_KO_data, peaks=GRanges_TSS_DE_genes, score = DBA_SCORE_RPKM)

dba.peakset(WT_KO_TSS_counts,bRetrieve = TRUE)

GRanges object with 94 ranges and 5 metadata columns:
     seqnames                 ranges strand |              CWS37               CWS38              CWS41              CSJ87              CSJ89
        <Rle>              <IRanges>  <Rle> |          <numeric>           <numeric>          <numeric>          <numeric>          <numeric>
   1     chr1 [186621791, 186623791]      * |   1.28321078430491    1.58536316777965   1.75570845030605   13.3095282913037   3.57979720307908
   2     chr1 [190117034, 190119034]      * |  0.203874610590499   0.341754934371661  0.390157433401345   4.27806266506192   1.07112042296854
   3     chr1 [193220640, 193222640]      * |  0.371771348723852   0.360741319614531  0.471440232026626 0.0950680592235982   1.12749518207215
   4     chr1 [194937820, 194939820]      * |   5.01291689569581    4.62318480663886   7.70560930967657 0.0950680592235982   8.00521579271227
   5    chr10 [ 90997491,  90999491]      * |  0.563653335161969   0.958812454764937   0.69903206817741 0.0950680592235982   2.11405346638528
  ..      ...                    ...    ... .                ...                 ...                ...                ...                ...
  90     chrX [128489858, 128491858]      * |  0.179889362285735     0.1898638524287 0.0162565597250561 0.0950680592235982 0.0281873795518038
  91     chrY [ 46054983,  46056983]      * |  0.239852483047646   0.218343430293005 0.0162565597250561 0.0950680592235982 0.0281873795518038
  92     chrY [ 86200359,  86202359]      * | 0.0119926241523823 0.00949319262143502 0.0162565597250561 0.0950680592235982 0.0281873795518038
  93     chrY [ 86767848,  86769848]      * | 0.0119926241523823 0.00949319262143502 0.0162565597250561 0.0950680592235982 0.0281873795518038
  94     chrY [117265245, 117267245]      * | 0.0119926241523823 0.00949319262143502 0.0162565597250561 0.0950680592235982 0.0281873795518038

 

Thanks in advance,

 

Pau.

 

ADD COMMENTlink modified 6 months ago by Rory Stark2.6k • written 7 months ago by pau.rodriguez.sodupe0
0
gravatar for Rory Stark
6 months ago by
Rory Stark2.6k
CRUK, Cambridge, UK
Rory Stark2.6k wrote:

Do any of the ranges in GRanges_TSS_DE_genes overlap? DiffBind will merge any overlapping ranges. You can check for these using eg. findOverlaps.

If you'd like, you can send me GRanges_TSS_DE_genes and I can check to see how DiffBind is handling it.

ADD COMMENTlink written 6 months ago by Rory Stark2.6k
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