transcript not found

[AndrewLangvt]

Hello SUPPA crew!

I've been having some issues with getting the IDs to match up between the abundance file and my ioe file. Here's what I'm working with...

GCF_000337935.1_Cliv_1.0_genomic.gtf

NW_004973172.1  Gnomon  exon    1051    1236    .       +       .       gene_id "102083463"; transcript_id "XM_005497964.1";
NW_004973172.1  Gnomon  exon    2168    2472    .       +       .       gene_id "102083463"; transcript_id "XM_005497964.1";
NW_004973172.1  Gnomon  exon    8449    8571    .       +       .       gene_id "102083654"; transcript_id "XM_005497965.2";
NW_004973172.1  Gnomon  exon    23499   23757   .       +       .       gene_id "102083654"; transcript_id "XM_005497965.2";
NW_004973172.1  Gnomon  exon    51968   52206   .       +       .       gene_id "102083654"; transcript_id "XM_005497965.2";
NW_004973173.1  Gnomon  exon    4340    4527    .       +       .       gene_id "102084630"; transcript_id "XM_013372080.1";
NW_004973173.1  Gnomon  exon    11701   11835   .       +       .       gene_id "102084630"; transcript_id "XM_013372080.1";
NW_004973173.1  Gnomon  exon    12814   12938   .       +       .       gene_id "102084630"; transcript_id "XM_013372080.1";
NW_004973173.1  Gnomon  exon    13939   13952   .       +       .       gene_id "102084630"; transcript_id "XM_013372080.1";
NW_004973173.1  Gnomon  exon    14071   14155   .       +       .       gene_id "102084630"; transcript_id "XM_013372080.1";

ColLiv.allevents.ioe

NW_004973175.1  102087983       102087983;A3:NW_004973175.1:737370-738407:737370-738412:+       XM_005497995.2  XM_005497995.2,XM_005497992.2,XM_005497991.2
NW_004973178.1  102096347       102096347;A3:NW_004973178.1:37267-37489:37262-37489:-   XM_005498042.2  XM_005498042.2,XM_005498043.1
NW_004973178.1  102085005       102085005;A3:NW_004973178.1:52168-52854:52162-52854:-   XM_013368009.1  XM_013368009.1,XM_013368029.1
NW_004973179.1  102089602       102089602;A3:NW_004973179.1:1338172-1340967:1337719-1340967:-   XM_005498092.2  XM_005498092.2,XM_005498095.2,XM_005498097.2,XM_005498094.2,XM_005498096.2,XM_005498093.2,XM_005498091.2
NW_004973180.1  102097913       102097913;A3:NW_004973180.1:168779-169727:168776-169727:-       XM_005498138.1  XM_005498138.1,XM_005498139.1
NW_004973180.1  102083465       102083465;A3:NW_004973180.1:778255-780588:777670-780588:-       XM_013371569.1,XM_013371543.1,XM_013371398.1,XM_013371513.1,XM_013371496.1,XM_013371472.1,XM_013371443.1,XM_013371419.1,XM_013371368.1  XM_013371569.1,
NW_004973180.1  102083465       102083465;A3:NW_004973180.1:762515-775475:762512-775475:-       XM_013371496.1,XM_005498145.1   XM_013371496.1,XM_005498145.1,XM_013371513.1
NW_004973182.1  102084883       102084883;A3:NW_004973182.1:3522841-3523368:3522841-3523377:+   XM_005498239.2,XM_005498238.2,XM_013366718.1    XM_005498239.2,XM_005498238.2,XM_013366718.1,XM_005498237.2,XM_005498240.2
NW_004973182.1  102092693       102092693;A3:NW_004973182.1:6460037-6462036:6460037-6462039:+   XM_005498283.1,XM_005498284.1   XM_005498283.1,XM_005498284.1,XM_013366681.1
NW_004973182.1  102089790       102089790;A3:NW_004973182.1:643756-643982:643698-643982:-       XM_013366666.1,XM_005498179.1   XM_013366666.1,XM_005498179.1,XM_013366668.1
NW_004973182.1  102098451       102098451;A3:NW_004973182.1:2226212-2226688:2226209-2226688:-   XM_013366754.1  XM_013366754.1,XM_013366751.1

v1_iso_TPM.txt

blu7-x_female_gonad_stress_v1   blu7-x_female_hypothalamus_stress_v1    blu7-x_female_pituitary_stress_v1       blu-o-x-ATLAS_female_gonad_control_v1   blu-o-x-ATLAS_female_hypothalamus_control_v1    blu-o-x-ATLAS_female_pituitary_control_v1       g105-x_male_gonad_stress_v1     g105-x_male_hypothalamus_stress_v1      g105-x_male_pituitary_stress_v1 L-Blu13_male_gonad_control_v1   L-Blu13_male_hypothalamus_control_v1    L-Blu13_male_pituitary_control_v1       L-G107_male_gonad_control_v1    L-G107_male_hypothalamus_control_v1     L-G107_male_pituitary_control_v1        L-O116_male_gonad_stress_v1     L-O116_male_hypothalamus_stress_v1      L-O116_male_pituitary_stress_v1 L-R2_male_gonad_stress_v1       L-R2_male_hypothalamus_stress_v1        L-R2_male_pituitary_stress_v1   L-R3_male_gonad_control_v1      L-R3_male_hypothalamus_control_v1       L-R3_male_pituitary_control_v1  r6-x_female_gonad_control_v1    r6-x_female_hypothalamus_control_v1     r6-x_female_pituitary_control_v1        R-Blu12_female_gonad_stress_v1  R-Blu12_female_hypothalamus_stress_v1   R-Blu12_female_pituitary_stress_v1      R-W44_female_gonad_control_v1   R-W44_female_hypothalamus_control_v1    R-W44_female_pituitary_control_v1       R-W7_female_gonad_stress_v1     R-W7_female_hypothalamus_stress_v1      R-W7_female_pituitary_stress_v1
NM_001282808.1  0       1.57162 0       0.66475 0       0.220831        0       0.907605        0.906405        0       2.25501 0       0.415875        0       0       0       1.19995 0.990853        0.524462        0.535496        0       1.87365 0       0       2.10042 0       0       0.259625        1.2192  0       0.204883        0.381553        0       1.1309  0       0
NM_001282809.1  1.29073 1.2379  50.0972 0.268697        0       35.302  0.331408        0.926947        83.7728 0.735261        0       10.1895 0.663845        1.67197 24.4242 0       0.237119        20.4936 1.1076  0.162229        17.0305 0.993135        0.315786        55.6423 0       0       44.9443 0.388061        0.369715        29.4024 0.611911        0.327666        15.2097 0.107806        0       17.1811
NM_001282810.1  722.347 972.971 935.241 477.179 1102.49 762.617 107.541 774.884 1348.99 227.704 644.564 977.259 152.474 716.733 749.67  119.783 1572.66 947.73  162.487 750.441 933.566 85.087  684.716 957.027 1297.46 1000.88 1102.45 838.527 902.41  1045.09 1263.36 920.583 1164.87 1300.6  862.808 863.928
NM_001282811.1  33.2807 1328.19 3.86107 19.8139 223.623 0.508051        48.2987 1200.57 0       33.2902 660.107 0       27.1279 482.677 4.00297 0       1949.45 2.13059 27.23   547.382 6.60312 15.0452 1412.1  7.20179 14.7763 973.031 6.07347 26.9111 1047.95 8.98875 50.1585 1045.98 3.11808 5.75796 814.954 0
NM_001282812.1  7.96707 25.3233 5.50359 7.48515 5.63888 2.94712 0.201178        9.43736 4.35983 0.252396        24.0326 2.63242 0       10.897  2.74107 0       0.104697        10.692  0.242864        15.461  2.14724 0       6.72224 3.53525 12.185  16.1811 18.9998 4.93922 27.7648 6.04042 2.90592 3.12687 6.31379 1.9938  0.810314        6.18845
NM_001282813.1  0.442975        0.690355        13.2517 1.24213 8.18473 5.1655  9.87322 0.810658        11.7691 11.3146 0       19.5109 4.20998 0       5.36099 6.19408 0.370103        9.448   2.29165 0.81493 6.26402 11.0944 0       19.2161 0       0       31.2819 0.251072        0.897543        6.12253 0.375611        0.179238        14.0786 0.546195        0       5.30271
NM_001282814.1  4.00726 36.2563 2.89471 8.17849 20.6584 2.45488 17.4784 32.924  5.04972 11.8574 111.021 3.83462 9.76278 67.4373 3.44322 25.2416 4.01029 6.59979 13.9706 33.8739 4.68959 22.5148 37.5436 3.45249 3.30783 55.7678 6.49054 4.11995 40.5615 5.45875 4.35934 43.936  4.20431 3.48934 28.8335 1.01808
NM_001282815.1  17.325  12.2492 27.7845 12.2727 16.8283 13.7342 4.5869  23.9672 67.7658 4.84483 16.408  64.423  3.13273 19.7156 68.3625 2.56462 1.76188 62.4702 3.7879  29.167  58.7276 3.30422 7.85101 62.3308 6.99702 5.24579 38.0989 12.5421 10.4174 21.7266 10.1487 17.4345 19.2334 10.1115 12.1205 27.7903
NM_001282816.1  68.2551 121.745 105.4   68.7224 129.645 62.9095 92.6033 116.025 119.952 100.927 124.965 105.603 79.0448 133.861 82.9592 75.1333 113.585 110.076 83.6091 105.415 109.217 75.4153 113.578 119.154 45.5431 108.188 79.2438 57.7413 113.294 77.7973 32.4269 119.164 66.4777 69.8054 104.139 83.1719
NM_001282817.1  8.34595 1.65055 1.18683 2.67117 0       1.36009 3.03645 2.83289 1.61971 1.80269 0       1.3186  2.9495  0.807237        2.19773 1.75392 0       2.64997 2.87399 1.53462 0       1.48808 1.30741 1.13709 8.7492  8.86501 2.80919 4.3131  1.79954 2.42465 9.72328 1.2851  0       12.8626 0.553145        0
NM_001282818.1  0       0       0       0       0       0       0       0       0.301729        0       0       0       0.548696        1.98546 0.321877        0.365359        0.158749        0       0       0       2.17415 1.23488 0       0       0       0       0       0       0       0       0       0       0       0       0       0.94762
NM_001282819.1  12.3368 0       0       3.89595 0       0       1.54146 0.409267        0       1.71691 0       0       1.32878 0       0       1.50435 0       0.39388 1.47546 2.00523 0       2.49115 1.55422 0       50.1139 0       0       16.1181 0.679577        0       25.9584 0.242765        0       0       0.804466        0
NM_001282820.1  61.4997 2.77024 2.84495 34.1643 11.9293 0.470096        3.21776 12.8966 0       1.32484 11.4826 4.07571 2.00991 4.50486 2.57927 1.54119 5.85746 3.52275 1.98168 8.55367 1.75213 0       2.5772  6.44071 85.5255 1.36833 4.33828 192.328 4.22576 1.06496 184.302 16.1617 0.830061        67.7664 9.59892 2.28533
NM_001282821.1  0       0.547123        0       0       3.41916 0       15.4926 0.55032 0.63027 13.8689 3.90285 0       17.0333 0.819537        0       22.2942 0       0.268618        25.0199 0.281742        0       17.3363 0       0       0       0       0       0       1.56766 0       0.129165        0       0       0       0       0
NM_001282822.1  0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       0       3.62285 0       0       0       0       0       0       0       0.263681        0       0       0       0
NM_001282823.1  19.3793 1.87017 0.497018        5.07065 2.11047 1.42105 57.4257 1.40738 0       61.1133 0       1.1345  26.2796 0       3.27942 38.255  0.000425725     0.672164        48.4739 1.12974 1.20478 60.7452 0.608354        2.44581 6.85487 0       1.60292 9.12418 0.637802        0.971116        8.1099  1.53185 1.23369 16.67   0.364868        0.511185
NM_001282824.1  0.74587 0       0.567711        0       0       0       4.90536 0       0       0       0       0       0.695432        0       0       4.39999 0       0       1.39846 0       0       0       0       0       0       0       0       0       0       0       0       0.222912        0       0.363655        0       0
NM_001282825.1  0       0       3.28149 0       0.572221        0       0       0       0       0.591541        0       3.76795 0       0       2.05539 0       0.173325        0       0.241558        0       4.18911 0       0       3.39584 0       0       0       0.232903        0.73725 2.90115 0.457461        0.0897866       1.87047 0.318047        0       4.38529

when I run the psiPerEvent function of SUPPA, I get numerous errors, all indicating a transcript ID was not found in the "expression file".

INFO:psiCalculator:Buffering transcript expression levels.
INFO:lib.tools:File /pylon2/mc3bg6p/al2025/isoform/SUPPA/v1_iso_TPM.txt closed.
INFO:lib.tools:File /pylon2/mc3bg6p/al2025/isoform/SUPPA/ColLiv_V1_events/ColLiv.allevents.ioe opened in reading mode.
INFO:psiCalculator:Calculating PSI from the ioe file.
ERROR:psiCalculator:transcript XM_005497995.2 not found in the "expression file".
ERROR:psiCalculator:PSI not calculated for event 102087983;A3:NW_004973175.1:737370-738407:737370-738412:+.
ERROR:psiCalculator:transcript XM_005498042.2 not found in the "expression file".
ERROR:psiCalculator:PSI not calculated for event 102096347;A3:NW_004973178.1:37267-37489:37262-37489:-.
ERROR:psiCalculator:transcript XM_013368009.1 not found in the "expression file".
ERROR:psiCalculator:PSI not calculated for event 102085005;A3:NW_004973178.1:52168-52854:52162-52854:-.
ERROR:psiCalculator:transcript XM_005498092.2 not found in the "expression file".
ERROR:psiCalculator:PSI not calculated for event 102089602;A3:NW_004973179.1:1338172-1340967:1337719-1340967:-.
ERROR:psiCalculator:transcript XM_005498138.1 not found in the "expression file".
ERROR:psiCalculator:PSI not calculated for event 102097913;A3:NW_004973180.1:168779-169727:168776-169727:-.
ERROR:psiCalculator:transcript XM_013371569.1 not found in the "expression file".

I did invoke the -p flag when generating events, as these are RefSeq annotations. Is there something funky going on here? Or is it as simple, yet perhaps bizarre, as a subset of transcripts (~3.5k of ~20K) are present in the gtf, yet absent from the transcriptome? Any thoughts?

[EduEyras]

Hi Andy,

sorry for the delay in replying. I usually get emails telling me there is an issue, but I did not get this one.

The -p should redefine genes, but that should not affect the link between events<-->isoforms and between isoforms <--> TPM values.

Your error says that for some of the XM_... transcripts you don't have data in the TPM file. We see this generally when we have TPM data for only some chromosomes (e.g. in human we removed unassembled chromosomes or haplotypes), or for only some genes (if we e.g. removed pseudogenes). It could be that you're original GTF had more annotations than you have TPM values for.

Please let me know if this helps

Best

Eduardo

[AndrewLangvt]

Thanks for the reply Eduardo. Is there any way to have SUPPA ignore those annotations for which there are no TPM values? Or do I have to parse through the annotations file and remove all of those annotations?

[EduEyras]

I'm afraid you'll have to remove them from the annotation file. The warning is there to let you know that there is a discrepancy between the annotation and the expression file. You can simply ignore de warnings, unless you're redirecting STDERR to log your runs in a cluster and you're running thousands of them and you don't want to end up filling up the disk. Then best will be to remove those cases from the annotation. I hope it works for you. Best Eduardo

On Wed, Nov 1, 2017 at 6:51 PM, AndrewLangvt notifications@github.com wrote:

Thanks for the reply Eduardo. Is there any way to have SUPPA ignore those annotations for which there are no TPM values? Or do I have to parse through the annotations file and remove all of those annotations?

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-- Dr E Eyras

ICREA Research Professor Universitat Pompeu Fabra PRBB, Dr Aiguader 88 Tel: +34 93 316 0502 (ext 1502) E08003 Barcelona, Spain Fax: +34 93 316 0550

http://scholar.google.com/citations?user=LiojlGoAAAAJ http://www.researcherid.com/rid/L-1053-2014 http://regulatorygenomics.upf.edu/

[AndrewLangvt]

Will the analysis run unhindered by the warnings if I were to simply ignore them? I'm running AS analyses with a few different programs, so I would like to keep things as consistent as possible across all analyses. If removing the annotations from the GTF is the only way to successfully complete SUPPAs analysis, that would be the only course of action.

[EduEyras]

Oh I see. No, it won't influence the results. It's just a warning. The results should be ok for the cases where it found the data.

That's interesting. We've been also comparing various methods: rMATS, MAJIQ and DEXSeq. I'd be happy to share experiences. I'd be curious to know if you're testing others and what you're results show (if you can share it, of course).

best

Eduardo

On Wed, Nov 1, 2017 at 6:59 PM, AndrewLangvt notifications@github.com wrote:

Will the analysis run unhindered by the warnings if I were to simply ignore them? I'm running AS analyses with a few different programs, so I would like to keep things as consistent as possible across all analyses. If removing the annotations from the GTF is the only way to successfully complete SUPPAs analysis, that would be the only course of action.

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-- Dr E Eyras

ICREA Research Professor Universitat Pompeu Fabra PRBB, Dr Aiguader 88 Tel: +34 93 316 0502 (ext 1502) E08003 Barcelona, Spain Fax: +34 93 316 0550

http://scholar.google.com/citations?user=LiojlGoAAAAJ http://www.researcherid.com/rid/L-1053-2014 http://regulatorygenomics.upf.edu/

[AndrewLangvt]

Great! That will save me some time, and also maintain consistency. Thanks, again, for addressing this!

Yea, I'll keep you guys posted as things unfold a bit more. Currently I'm just running a subset of my data (36 rnaseq libraries, full dataset is 152) through SUPPA, JunctionSeq (heavily based on DEXSeq), and Whippet. Once I have a clearer picture of the agreement/disagreements between the three, I'd be happy to chat further and discuss!

Cheers

[EduEyras]

On Wed, Nov 1, 2017 at 7:12 PM, AndrewLangvt notifications@github.com wrote:

Great! That will save me some time, and also maintain consistency. Thanks, again, for addressing this!

Yea, I'll keep you guys posted as things unfold a bit more. Currently I'm just running a subset of my data (36 rnaseq libraries, full dataset is 152) through SUPPA, JunctionSeq (heavily based on DEXSeq), and Whippet. Once I have a clearer picture of the agreement/disagreements between the three, I'd be happy to chat further and discuss!

I'd be curious how to compare Whippet with SUPPA. When comparing with MAJIQ, we were selecting junctions, and using the junction with the highest posterior (the most confident) and assign it to the even (for SE events it would be the inclusion junction).

For JunctionSeq I'd imagine it would be something similar. For Whippet I don't know if it'll be simple to do.

best

E.

Cheers

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-- Dr E Eyras

ICREA Research Professor Universitat Pompeu Fabra PRBB, Dr Aiguader 88 Tel: +34 93 316 0502 (ext 1502) E08003 Barcelona, Spain Fax: +34 93 316 0550

http://scholar.google.com/citations?user=LiojlGoAAAAJ http://www.researcherid.com/rid/L-1053-2014 http://regulatorygenomics.upf.edu/

[Oliverfeudj]

Hello @EduEyras can you please help me with this issue? it says it for all transcripts and gives NA in psi file ERROR:psiCalculator:transcript ENSMUSG00000051285 not found in the "expression file". ERROR:psiCalculator:PSI not calculated for event ENSMUSG00000094337;SE:GL456354.1:83560-84521:85111-85765:-.

here is my expression file in tpm

my ioe file

when I grep ENSMUSG00000051285 in expression file

I have check if there are hidden spaces in the tab file but it seems okay

Thank you for your help

[EduEyras]

Hi These are gene ids But you need to quantify the transcript ids Best E

On Wed, 15 Mar 2023 at 04:58, Olivier Feudjio @.***> wrote:

Hello @EduEyras https://github.com/EduEyras can you please help me with this issue? it says it for all transcripts and gives NA in psi file ERROR:psiCalculator:transcript ENSMUSG00000051285 not found in the "expression file". ERROR:psiCalculator:PSI not calculated for event ENSMUSG00000094337;SE:GL456354.1:83560-84521:85111-85765:-.

here is my expression file in tpm [image: image] https://user-images.githubusercontent.com/52349851/225257686-2bd15c7f-bc25-4ba9-aaba-29c69bb9d27f.png

my ioe file [image: image] https://user-images.githubusercontent.com/52349851/225257862-746d610b-54f7-4fd8-9951-c883a099da3f.png

when I grep ENSMUSG00000051285 in expression file [image: image] https://user-images.githubusercontent.com/52349851/225257992-d67966a0-4059-45f0-bf88-2c90438f32d0.png

I have check if there are hidden spaces in the tab file but it seems okay

Thank you for your help

— Reply to this email directly, view it on GitHub https://github.com/comprna/SUPPA/issues/10#issuecomment-1469603829, or unsubscribe https://github.com/notifications/unsubscribe-auth/ADCZKB2WQXB3NNXNHFIIOD3W4GACXANCNFSM4EAT6UUQ . You are receiving this because you were mentioned.Message ID: @.***>

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[Oliverfeudj]

Thank you for your reply @EduEyras Actually, these are transcripts ids, I just remove some extensions this is how the original file looks like:

If you have an example of what you mean by transcript ids, please show me

Thank you!

[EduEyras]

Hi, sorry, I am confused your expression file seemed to use gene ids (ENSMUSG....) rather than transcript ids (ENSMUST....)

E.

On Wed, 15 Mar 2023 at 11:11, Olivier Feudjio @.***> wrote:

Thank you for your reply @EduEyras https://github.com/EduEyras Actually, these are transcripts ids, I just remove some extensions this is how the original file looks like: [image: image] https://user-images.githubusercontent.com/52349851/225353426-824b6828-6d1f-45c8-a9aa-834dc1f60309.png

If you have an example of what you mean by transcript ids, please show me

Thank you!

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[Oliverfeudj]

Yeah it's my mistake, I used ENSMUST.... but still got the same errors, for instance: ERROR:psiCalculator:transcript ENSMUST00000236635 not found in the "expression file". ERROR:psiCalculator:PSI not calculated for event ENSMUSG00000033768;SE:19:6505310-6513854:6513877-6522288:+.

Thank you for dedicating your time to this issue

[EduEyras]

In this case, it could be that ENSMUST00000236635 is not expressed, All the transcripts that are in the annotation and hence in the ioe file, but are not expressed, will give you that error. It should be normal. Do you get PSIs for the other events? best Eduardo

On Wed, 15 Mar 2023 at 11:49, Olivier Feudjio @.***> wrote:

Yeah it's my mistake, I used ENSMUST.... but still got the same errors, for instance: ERROR:psiCalculator:transcript ENSMUST00000236635 not found in the "expression file". ERROR:psiCalculator:PSI not calculated for event ENSMUSG00000033768;SE:19:6505310-6513854:6513877-6522288:+.

Thank you for dedicating your time to this issue

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[Oliverfeudj]

That is the issue, I got NA for all the transcripts

no psi values best

[EduEyras]

One possibility is a formatting problem in the input files, like hidden characters at the end of the line, or a missing header. E.

On Wed, 15 Mar 2023 at 11:59, Olivier Feudjio @.***> wrote:

That is the issue, I got NA for all the transcripts

no psi values best

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[Oliverfeudj]

The header is not missing and I don't think there are hidden characters at the end of the file Please find attached the two expression files I am using for my analysis if you don't mind.

Thank you expression files.zip

[EduEyras]

Could you please send me your command line and a sample of your .ioe file

thanks

E.

On Wed, 15 Mar 2023 at 14:21, Olivier Feudjio @.***> wrote:

The header is not missing and I don't think there are hidden characters at the end of the file Please find attached the two expression files I am using for my analysis if you don't mind.

Thank you expression files.zip https://github.com/comprna/SUPPA/files/10983398/expression.files.zip

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[Oliverfeudj]

Yes sure, thank you mm10_SE_strict.zip

[Oliverfeudj]

And here is the command line: suppa.py psiPerEvent --ioe-file mm10_SE_strict.ioe --expression-file spc.tsv --output-file ./spc_SE

[Oliverfeudj]

Hello @EduEyras, Just checking if you have had a little time to look at my request, please?

Thank you