PRiano-bio commented 2 months ago

I sequenced with Nanopore (latest 10.4 flowcell) DNA samples that resulted positive for BrdU and EdU incorporation via immunoblot. After running DNAscent 4.0.3 via Singularity, the output of forksense consists in one single read with just 0s for EdU and BrdU value. Moreover in the terminal output, both DNAscent detect and DNAscent forksense have 100% of "failed". Here below I reported the reduced version of the output files and the output of the terminal.

TERMINAL OUTPUT: Even if the terminal output reported is truncated, there were no value other that "failed".

Processing sample: Test
Indexing BAM file...
All steps completed successfully for sample Test
Running DNAscent index step...
[========>                          ]  25%   1/4   0hr 0min 0sec  
[=================>                 ]  50%   2/4   0hr 0min 0sec  
[==========================>        ]  75%   3/4   0hr 0min 0sec  
[===================================] 100%   4/4   0hr 0min 0sec  
Running DNAscent detect step...
Loading DNAscent index... ok.
Importing reference... ok.
Opening bam file... ok.
Scanning bam file...ok.
[>                                  ]   0%      0/3639   0hr 0min 0sec  failed:     0  
[>                                  ]   0%      0/3639   0hr 0min 0sec  failed:     0  
[>                                  ]   0%      0/3639   0hr 0min 0sec  failed:     0  
[...]
[==================================>]  99%   3632/3639   0hr 0min 2sec  failed:   471  
[==================================>]  99%   3632/3639   0hr 0min 2sec  failed:   471  
[===================================] 100%   3639/3639   0hr 0min 0sec  failed:   478  
Running DNAscent forksense step...
[>                                  ]   0%      1/3161   0hr 0min 0sec  failed:     0  
[>                                  ]   0%      2/3161   0hr 0min 0sec  failed:     1  
[>                                  ]   0%      3/3161   0hr 0min 1sec  failed:     2  
[...]
[==================================>]  99%   3159/3161   0hr 0min 0sec  failed:  3031  
[==================================>]  99%   3160/3161   0hr 0min 0sec  failed:  3032  
[===================================] 100%   3161/3161   0hr 0min 0sec  failed:  3033  
Finished processing sample: Test
All samples processed. Job finished at Mon Sep  9 02:29:50 PM CEST 2024

OUTPUT.DETECT :

#Alignment /storage/home/user/dnascent-output/Test.sorted.bam
#Genome /storage/home/user/reference.fasta
#Index /storage/home/user/dnascent-output/Test.index.dnascent
#Threads 1
#Compute GPU
#Mode CNN
#MappingQuality 20
#MappingLength 1000
#SystemStartTime 09/09/2024 14:10:45
#Software /app/DNAscent/
#Version 4.0.3
#Commit 9f3ef31b58cb12a35795d31ebd42b97e6d8de890
>14864159-7402-4a4c-976a-ae5326137b92 reference 0 2397 fwd
10  0.064936    0.038356    GTGCTGCAA
20  0.127683    0.071554    GCGATTAAG
21  0.099561    0.048271    CGATTAAGT
25  0.129081    0.079023    TAAGTTGGG
26  0.117107    0.092562    AAGTTGGGT
[...]
2390    0.016510    0.034979    TTGTTTAGT
2391    0.019896    0.054742    TGTTTAGTT

TERMINAL OUTPUT:


2024-09-09 14:29:44.603341: I tensorflow/stream_executor/platform/default/dso_loader.cc:49] Successfully opened dynamic library libcudart.so.11.0
Estimated fraction of BrdU substitution in BrdU-positive regions: 0.271571
Estimated BrdU substitution lower bound in BrdU-positive regions: 0.189627
Estimated fraction of EdU substitution in EdU-positive regions: 0.127939
Estimated EdU substitution lower bound in EdU-positive regions: 0.0681818


2. **OUTPUT.FORKSENSE:**
Even if the file reported is truncated, no value different from 0 are present in the file.

DetectFile /storage/home/user/dnascent-output/Test.output.detect

Threads 1

Compute CPU

SystemStartTime 09/09/2024 14:29:47

Software /app/DNAscent/

Version 4.0.3

Commit 9f3ef31b58cb12a35795d31ebd42b97e6d8de890

EstimatedRegionBrdU 0.271571

EstimatedRegionEdU 0.127939

0b022afb-d4e1-4974-8dee-355a6ad0f4e2 Telnn.lin 0 5211 rev 4 0 0 13 0 0 14 0 0 [...] 7922 0 0 7937 0 0 7944 0 0

MBoemo commented 2 months ago

Hard to say - there are quite a few reasons this could happen based on your experimental setup. I'd need a lot more information on what you did on the wet lab side. Feel free to Email me at mb915@cam.cam.ac.uk to discuss if that's easier.