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open2c / coolpuppy

A versatile tool to perform pile-up analysis on Hi-C data in .cool format.
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Pileup for telomeres concerning trans chromosomal interactions #150

Closed Brahmandam-Gayatri closed 3 months ago

Brahmandam-Gayatri commented 3 months ago

Hello,

Thank you for developing coolpuppy for pileup analysis. I want to plot an aggregate heatmap for yeast data using telomeric coordinates and I am unable to do so.

chrom start end chrIX 1 7784 chrV 1 6473 chrXIII 1 6344 chrXVI 1 7223 chrIII 1 1098 chrVI 1 5530 chrXII 1 12085 chrVIII 1 5505 chrXV 1 847 chrII 1 6608 chrXIV 1 7428 chrI 1 801 chrVII 1 781 chrX 1 7767 chrXI 1 807 chrIV 1 904

In general, the plot looks similar to the aggregate plot of centromeres, but here the aggregate signal is observed at the corners in place of center. Any suggestions are highly appreciated!!

Phlya commented 3 months ago

Please provide more details - what have you tried, what were the results?

Brahmandam-Gayatri commented 3 months ago

I tried using cooltools, but not sure how to proceed with coolpuppy. Here is my code:

clr1_10kb = cooler.Cooler('/path/to/file/sample1.mcool::/resolutions/10000')

sacCer3_chromsizes = bioframe.fetch_chromsizes('sacCer3')
sacCer3_cens = pd.read_csv('/path/to/file/centromere.txt', sep='\t')

Read telomere coordinates:

tel_yeast = pd.read_table('/path/to/file/telomere_left_yeast.bed', sep='\t').query(f'chrom in {clr1_10kb.chromnames}')
tel_yeast['mid'] = (tel_yeast.end+tel_yeast.start)//2
tel_yeast.head()

chrom   start   end mid
0   chrIX   1   7784    3892
1   chrV    1   6473    3237
2   chrXIII 1   6344    3172
3   chrXVI  1   7223    3612
4   chrIII  1   1098    549

expected_yeast = cooltools.expected_cis(clr1_10kb, nproc=2, chunksize=1_000)

expected_yeast

region1 region2 dist    n_valid count.sum   balanced.sum    count.avg   balanced.avg    balanced.avg.smoothed   balanced.avg.smoothed.agg
0   chrM    chrM    0   9   NaN NaN NaN NaN NaN NaN
1   chrM    chrM    1   8   NaN NaN NaN NaN 0.000497    0.000639
2   chrM    chrM    2   7   48293.0 0.372054    6899.000000 0.053151    0.052541    0.056308
3   chrM    chrM    3   6   36136.0 0.321482    6022.666667 0.053580    0.048051    0.037760
4   chrM    chrM    4   5   22451.0 0.167468    4490.200000 0.033494    0.037567    0.026177
... ... ... ... ... ... ... ... ... ... ...
1220    chrIV   chrIV   149 4   19.0    0.003011    4.750000    0.000753    0.000521    0.000535
1221    chrIV   chrIV   150 3   3.0 0.000386    1.000000    0.000129    0.000522    0.000536
1222    chrIV   chrIV   151 2   4.0 0.000706    2.000000    0.000353    0.000524    0.000537
1223    chrIV   chrIV   152 1   121.0   0.019644    121.000000  0.019644    0.000525    0.000538
1224    chrIV   chrIV   153 0   0.0 0.000000    NaN NaN 0.000526    0.000538
1225 rows × 10 columns

paired_sites = bioframe.pair_by_distance(tel_yeast, min_sep=200, max_sep=1000, suffixes=('1', '2'))
paired_sites.loc[:, 'mid1'] = (paired_sites['start1'] + paired_sites['end1'])//2
paired_sites
chrom1  start1  end1    mid1    chrom2  start2  end2    mid2  **#The paired_sites dataframe is empty**

I tried doing this to generate aggregate plot for off-diagonal pileup, as documented here: https://cooltools.readthedocs.io/en/latest/notebooks/pileup_CTCF.html

But as I could not get the plot, I went through issues of cooltools so as to raise a new issue (https://github.com/open2c/cooltools/issues/491) and was redirected to coolpuppy from https://github.com/open2c/cooltools/issues/491#issuecomment-1920075175

Hence, I went through the docs of https://coolpuppy.readthedocs.io/en/latest/Examples/Walkthrough_API.html#trans-inter-chromosomal-pileups, but I am still not sure how to plot for telomeric regions.

Phlya commented 3 months ago

Well, since your regions are on the edge of the chromosome, you need to basically just pileup all chromosome starts. So You can create a bed file with the first e.g. 100 kbp regions of each chromosome chrom start end chrIX 1 100000 ... etc.

Then use coolpup.py pileup with --trans and without rescaling.

Brahmandam-Gayatri commented 3 months ago

Apologies if I sound naive, but how to create the bed file?

Phlya commented 3 months ago

It's a simple tab-delimited file with three columns (chrom, start, end). You can use pandas, a text editor, or a spreadsheet editor.

Phlya commented 3 months ago

Or you can keep using the python API, then you can modify the tel_yeast dataframe

Brahmandam-Gayatri commented 3 months ago

So I need to just save the telomere coordinates as a bed file and input that along with my mcool file to coolpup.py pileup to get the plot using command line?

Brahmandam-Gayatri commented 3 months ago

This is how the sacCer3_cens data looks like:

chrom   start   end mid
0   chrI    151465  151582  151524
1   chrII   238207  238323  238265
2   chrIII  114385  114501  114443
3   chrIV   449711  449821  449766
4   chrV    151987  152104  152046
5   chrVI   148510  148627  148569
6   chrVII  496920  497038  496979
7   chrVIII 105586  105703  105645
8   chrIX   355629  355745  355687
9   chrX    436307  436425  436366
10  chrXI   440129  440246  440188
11  chrXII  150828  150947  150888
12  chrXIII 268031  268149  268090
13  chrXIV  628758  628875  628817
14  chrXV   326584  326702  326643
15  chrXVI  555957  556073  556015
Phlya commented 3 months ago

Sort of - actually there is a trick... you have to provide a file that is shifted by a certain distance from the end of the chromosome (e.g. 100 kb), and use the same distance as the --flank argument. So, small correction: It should look like this: chrom start end chrIX 90000 110000 ...

And use coolpup.py {cool_file} {shifted_tels.bed} --flank 100000 --trans --expected {expected_trans_file} -o {pileup.clp} And then plotpup.py should plot the result

Brahmandam-Gayatri commented 3 months ago

Should I input the same coordinates for all chromosomes or change according to the chromosome? For eg., chrII and chrIII have different coordinates, so would it be fine if I specify as 90000 and 110000 for both?

chrII   238207  238323  
chrIII  114385  114501
Phlya commented 3 months ago

Before you showed coordinates that were all very small and close to 0, why are they much bigger now?

Brahmandam-Gayatri commented 3 months ago

These are centromeric coordinates.

chrII   238207  238323  
chrIII  114385  114501

I showed the left arm telomeric coordinates at the start of my post

chrIII 1 1098
chrVI 1 5530
chrXII 1 12085
chrVIII 1 5505
chrXV 1 847
chrII 1 6608
Phlya commented 3 months ago

But you want to pileup telomeres?

Brahmandam-Gayatri commented 3 months ago

Yes. I want the pileup for telomeres of both left and right arms of the chromosomes. But to plot, I am only doing with left arm first.

Phlya commented 3 months ago

Yeah then just pileup around a fixed distance shifted from 0, e.g. 100 kb or smth... I'm not familiar with yeast data, not sure what would be the best distance. Then you can pileup the right ones the same way, shift from the end of each chromosome by a fixed distance

Brahmandam-Gayatri commented 3 months ago

I am getting the following error:

coolpup.py /path/to/file/sample1.mcool::/resolutions/10000 shifted_tels.bed --flank 100000 --trans --expected expected_trans.tsv -o pileup.clp
/usr/lib/python3/dist-packages/scipy/__init__.py:146: UserWarning: A NumPy version >=1.17.3 and <1.25.0 is required for this version of SciPy (detected version 1.26.4
  warnings.warn(f"A NumPy version >={np_minversion} and <{np_maxversion}"
Traceback (most recent call last):
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 298, in _is_compatible_trans_expected
    _ = _is_expected(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 152, in _is_expected
    raise e
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 125, in _is_expected
    raise ValueError(f"Values in {grouping_columns} columns must be unique")
ValueError: Values in ['region1', 'region2'] columns must be unique

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/io.py", line 327, in read_expected_from_file
    _ = is_valid_expected(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 391, in is_valid_expected
    return _is_compatible_trans_expected(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 340, in _is_compatible_trans_expected
    raise e
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 305, in _is_compatible_trans_expected
    raise ValueError("expected_df does not look like trans-expected") from e
ValueError: expected_df does not look like trans-expected

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "/home/catg1/.local/bin/coolpup.py", line 8, in <module>
    sys.exit(main())
  File "/home/catg1/.local/lib/python3.10/site-packages/coolpuppy/CLI.py", line 493, in main
    expected = io.read_expected_from_file(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/io.py", line 336, in read_expected_from_file
    raise ValueError(
ValueError: Input expected file does not match the schema
Expected must be tab-separated file with a header

The file expected_trans.tsv contains the following:

<!DOCTYPE html>

<html>
<head>

    <title></title>
    <meta name="generator" content="LibreOffice 7.3.7.2 (Linux)"/>
    <style type="text/css">
        body,div,table,thead,tbody,tfoot,tr,th,td,p { font-family:"Liberation Sans"; font-size:x-small }
        a.comment-indicator:hover + comment { background:#ffd; position:absolute; display:block; border:1px solid black; padding:0.5em;  } 
        a.comment-indicator { background:red; display:inline-block; border:1px solid black; width:0.5em; height:0.5em;  } 
        comment { display:none;  } 
    </style>

</head>

<body>

region1 | region2 | dist | n_valid | count.sum | balanced.sum | count.avg | balanced.avg | balanced.avg.smoothed | balanced.avg.smoothed.agg
-- | -- | -- | -- | -- | -- | -- | -- | -- | --
chrM | chrM | 0 | 9 |   |   |   |   |   |  
chrM | chrM | 1 | 8 |   |   |   |   | 0.000496669227937875 | 0.00063942972823703
chrM | chrM | 2 | 7 | 48293 | 0.372053637479411 | 6899 | 0.0531505196399159 | 0.0525410493153696 | 0.0563079144204046
chrM | chrM | 3 | 6 | 36136 | 0.321481934581842 | 6022.66666666667 | 0.053580322430307 | 0.0480513547631135 | 0.0377598171583264
chrM | chrM | 4 | 5 | 22451 | 0.167467893069907 | 4490.2 | 0.0334935786139813 | 0.0375673781338824 | 0.0261766749716914
chrM | chrM | 5 | 4 | 14185 | 0.11236548714211 | 3546.25 | 0.0280913717855276 | 0.0360963830657584 | 0.018969640466925
chrM | chrM | 6 | 3 | 11203 | 0.0887893943518971 | 3734.33333333333 | 0.0295964647839657 | 0.0491267465142774 | 0.0142832725033798
chrM | chrM | 7 | 2 | 10225 | 0.0805744713985754 | 5112.5 | 0.0402872356992877 | 0.0752928403921177 | 0.0110845475380622
chrM | chrM | 8 | 1 | 44000 | 0.377630196055131 | 44000 | 0.377630196055131 | 0.10876732008745 | 0.00880629387117672
chrIX | chrIX | 0 | 42 |   |   |   |   |   |  
chrIX | chrIX | 1 | 41 |   |   |   |   | 0.000587566452703396 | 0.00063942972823703
chrIX | chrIX | 2 | 40 | 31083 | 2.25873209324364 | 777.075 | 0.056468302331091 | 0.0522900364541279 | 0.0563079144204046
chrIX | chrIX | 3 | 39 | 19380 | 1.41740182842212 | 496.923076923077 | 0.0363436366262083 | 0.0346470550700121 | 0.0377598171583264
chrIX | chrIX | 4 | 38 | 12546 | 0.909496302874006 | 330.157894736842 | 0.0239341132335265 | 0.0234776546783782 | 0.0261766749716914
chrIX | chrIX | 5 | 37 | 8769 | 0.636014010291257 | 237 | 0.0171895678457097 | 0.0167191559864385 | 0.018969640466925
chrIX | chrIX | 6 | 36 | 6230 | 0.453913668018458 | 173.055555555556 | 0.0126087130005127 | 0.0123988360898993 | 0.0142832725033798
chrIX | chrIX | 7 | 35 | 4577 | 0.330308018504265 | 130.771428571429 | 0.00943737195726473 | 0.00947597931387518 | 0.0110845475380622
chrIX | chrIX | 8 | 34 | 3458 | 0.250111089468336 | 101.705882352941 | 0.00735620851377459 | 0.00743528836833192 | 0.00880629387117672
chrIX | chrIX | 9 | 33 | 2686 | 0.196342671245793 | 81.3939393939394 | 0.00594977791653917 | 0.00597241658428364 | 0.00712865424620211
chrIX | chrIX | 10 | 32 | 2079 | 0.15089710731848 | 64.96875 | 0.0047155346037025 | 0.00489941668906075 | 0.00586938932489326
chrIX | chrIX | 11 | 31 | 1683 | 0.119554717638327 | 54.2903225806452 | 0.00385660379478475 | 0.00409746125172262 | 0.00491551082671522
chrIX | chrIX | 12 | 30 | 1428 | 0.101074284498914 | 47.6 | 0.00336914281663046 | 0.00348811011483907 | 0.00418855820128917
chrIX | chrIX | 13 | 29 | 1142 | 0.0810218250968101 | 39.3793103448276 | 0.00279385603782104 | 0.00301770972112611 | 0.00363010272398591
chrIX | chrIX | 14 | 28 | 1021 | 0.0725704580713719 | 36.4642857142857 | 0.00259180207397757 | 0.00264889881396591 | 0.00319570812585238
chrIX | chrIX | 15 | 27 | 825 | 0.0586249749229456 | 30.5555555555556 | 0.0021712953675165 | 0.00235538487639591 | 0.00285180404455236
chrIX | chrIX | 16 | 26 | 687 | 0.0502465934841103 | 26.4230769230769 | 0.0019325612878504 | 0.00211854523421107 | 0.00257367476271558
chrIX | chrIX | 17 | 25 | 623 | 0.045339101246096 | 24.92 | 0.00181356404984384 | 0.00192505750135856 | 0.00234370630074062
chrIX | chrIX | 18 | 24 | 526 | 0.0381323243496206 | 21.9166666666667 | 0.00158884684790086 | 0.00176522448628715 | 0.0021497042831649
chrIX | chrIX | 19 | 23 | 507 | 0.0371118989933197 | 22.0434782608696 | 0.00161356082579651 | 0.00163192587023838 | 0.00198345183312927
chrIX | chrIX | 20 | 22 | 425 | 0.0310489700985334 | 19.3181818181818 | 0.00141131682266061 | 0.00151987608905784 | 0.0018394528675716
chrIX | chrIX | 21 | 21 | 382 | 0.0283030714115839 | 18.1904761904762 | 0.00134776530531352 | 0.00142505626400995 | 0.00171391190010117
chrIX | chrIX | 22 | 20 | 303 | 0.0215487693655638 | 15.15 | 0.00107743846827819 | 0.00134439350944602 | 0.00160408664679366
chrIX | chrIX | 23 | 19 | 294 | 0.0215262241712543 | 15.4736842105263 | 0.00113295916690812 | 0.00127548704655054 | 0.00150783719102629
chrIX | chrIX | 24 | 18 | 271 | 0.0198055954959348 | 15.0555555555556 | 0.00110031086088527 | 0.00121643777837355 | 0.00142338597723856
chrIX | chrIX | 25 | 17 | 240 | 0.0181570700365712 | 14.1176470588235 | 0.00106806294332772 | 0.00116570557816471 | 0.00134918031338564

</body>

</html>

And the shifted_tels.bed has the following coordinates:

chrom   start   end
chrIX   9000    110000
chrV    9000    110000
chrXIII 9000    110000
chrXVI  9000    110000
chrIII  9000    110000
chrVI   9000    110000
chrXII  9000    110000
chrVIII 9000    110000
chrXV   9000    110000
chrII   9000    110000
chrXIV  9000    110000
chrI    9000    110000
chrVII  9000    110000
chrX    9000    110000
chrXI   9000    110000
chrIV   9000    110000
Brahmandam-Gayatri commented 3 months ago

I computed the trans expected and reran the command. But still the same error:

This is the trans file output:

region1 region2 n_valid count.sum   balanced.sum    count.avg   balanced.avg
0   chrM    chrIX   378 9299.0  0.206373    24.600529   0.000546
1   chrM    chrV    504 11856.0 0.281875    23.523810   0.000559
2   chrM    chrXIII 783 19297.0 0.440791    24.644955   0.000563
3   chrM    chrXVI  801 18673.0 0.444724    23.312110   0.000555
4   chrM    chrIII  288 6642.0  0.147612    23.062500   0.000513
... ... ... ... ... ... ... ...
131 chrVII  chrXI   7102    45664.0 3.478307    6.429738    0.000490
132 chrVII  chrIV   15688   97194.0 7.622758    6.195436    0.000486
133 chrX    chrXI   4757    33967.0 2.578139    7.140425    0.000542
134 chrX    chrIV   10508   62620.0 5.130233    5.959269    0.000488
135 chrXI   chrIV   9916    57778.0 4.469080    5.826745    0.000451
136 rows × 7 columns

Error:

/usr/lib/python3/dist-packages/scipy/__init__.py:146: UserWarning: A NumPy version >=1.17.3 and <1.25.0 is required for this version of SciPy (detected version 1.26.4
  warnings.warn(f"A NumPy version >={np_minversion} and <{np_maxversion}"
Traceback (most recent call last):
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 298, in _is_compatible_trans_expected
    _ = _is_expected(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 152, in _is_expected
    raise e
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 125, in _is_expected
    raise ValueError(f"Values in {grouping_columns} columns must be unique")
ValueError: Values in ['region1', 'region2'] columns must be unique

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/io.py", line 327, in read_expected_from_file
    _ = is_valid_expected(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 391, in is_valid_expected
    return _is_compatible_trans_expected(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 340, in _is_compatible_trans_expected
    raise e
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/checks.py", line 305, in _is_compatible_trans_expected
    raise ValueError("expected_df does not look like trans-expected") from e
ValueError: expected_df does not look like trans-expected

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "/home/catg1/.local/bin/coolpup.py", line 8, in <module>
    sys.exit(main())
  File "/home/catg1/.local/lib/python3.10/site-packages/coolpuppy/CLI.py", line 493, in main
    expected = io.read_expected_from_file(
  File "/home/catg1/.local/lib/python3.10/site-packages/cooltools/lib/io.py", line 336, in read_expected_from_file
    raise ValueError(
ValueError: Input expected file does not match the schema
Expected must be tab-separated file with a header

My mistake, I reran the command with proper file and it worked, but when I ran plotpup.py, it says there are nans or zero values present:

coolpup.py /path/to/file/sample1.mcool::/resolutions/10000 shifted_tels.bed --flank 100000 --trans --expected expected_trans.txt -o pileup.clp
/usr/lib/python3/dist-packages/scipy/__init__.py:146: UserWarning: A NumPy version >=1.17.3 and <1.25.0 is required for this version of SciPy (detected version 1.26.4
  warnings.warn(f"A NumPy version >={np_minversion} and <{np_maxversion}"
/home/catg1/.local/lib/python3.10/site-packages/coolpuppy/coolpup.py:2156: UserWarning: Ignoring maxdist when using trans
  CC = CoordCreator(
**INFO:coolpuppy:Total number of piled up windows: 0
INFO:coolpuppy:Saved output to pileup.clp**
plotpup.py --input_pups pileup.clp
/usr/lib/python3/dist-packages/scipy/__init__.py:146: UserWarning: A NumPy version >=1.17.3 and <1.25.0 is required for this version of SciPy (detected version 1.26.4
  warnings.warn(f"A NumPy version >={np_minversion} and <{np_maxversion}"
/home/catg1/.local/lib/python3.10/site-packages/coolpuppy/lib/numutils.py:79: RuntimeWarning: Mean of empty slice
  return np.nanmean(amap[c - n // 2 : c + n // 2 + 1, c - n // 2 : c + n // 2 + 1])
Traceback (most recent call last):
  File "/home/catg1/.local/bin/plotpup.py", line 8, in <module>
    sys.exit(main())
  File "/home/catg1/.local/lib/python3.10/site-packages/coolpuppy/plotpuppy_CLI.py", line 369, in main
    fg = plot(
  File "/home/catg1/.local/lib/python3.10/site-packages/coolpuppy/plotpup.py", line 735, in plot
    vmin, vmax = get_min_max(pupsdf["data"].values, vmin, vmax, sym=sym, scale=scale)
  File "/home/catg1/.local/lib/python3.10/site-packages/coolpuppy/plotpup.py", line 83, in get_min_max
    raise ValueError("Data only contains NaNs or zeros")
ValueError: Data only contains NaNs or zeros
Phlya commented 3 months ago

/usr/lib/python3/dist-packages/scipy/init.py:146: UserWarning: A NumPy version >=1.17.3 and <1.25.0 is required for this version of SciPy (detected version 1.26.4 warnings.warn(f"A NumPy version >={np_minversion} and <{np_maxversion}"

I wonder whether it can cause issues...

Can you share the shifted_tels.bed file?

Brahmandam-Gayatri commented 3 months ago

I will try and upgrade numpy version. Sure, here is the file: Since bed file format is not supported, I am sharing the google drive link: https://drive.google.com/file/d/1CHV_MUifsFywEsFsJjeB1XqGjKnDUyp5/view?usp=drive_link

Brahmandam-Gayatri commented 3 months ago

I downgraded numpy version but still no windows were generated, and thank you so much taking time out in helping me solve my query Phlya!!

Phlya commented 3 months ago

You have starts set to 9000, but it should be 90000. The idea is that the midpoint of each interval is at 100,000 - then with 100,000 flank you pileup starting from 0 and catch the telomere.

Phlya commented 3 months ago

As an alternative, you can set start to 0, end to e.g. 99,000, and then use --rescale --rescale-size 99 --rescale-flank 0 (you can do the arithmetic so it works correctly with the resolution that you want to use, considering rescale size has to be odd)

Brahmandam-Gayatri commented 3 months ago

Okay, I tried by changing the 9000 to 90000 and I did get two plots (attached herewith), I will try the second method and let you know, thanks again!! pup_sample2.pdf pup_sample1.pdf

Phlya commented 3 months ago

Perfect! No need to try the other method if this worked IMO, the first one should be faster and maybe a little more precise.

Brahmandam-Gayatri commented 3 months ago

Okay, thanks a lot, I will need to do a bit of trial and error for understanding if I am doing the analysis correctly, but I understood the approach. Have a great day ahead, I am closing this issue for now, if I have any further queries I will reopen this, hope that is fine with you!!

ratheraarif commented 1 month ago

@Brahmandam-Gayatri Hi Gayatri, did you figured this out. I am also having the same issue with the telomeric coordinates of yeast, instead of signal being aggregated at the centre, its aggregated at the corner like this:

image

If you solved this issue, can you please share it. I am not sure how to set the flank and how much to shift the coordinates from the beginning.

Thanks!