thread 'main' panicked at 'called `Option::unwrap()` on a `None` value', src/util.rs:1401:33

[Ci-TJ]

Hi. I just installed the Terminus by conda. I didn't get any results after I run terminus with "group". Unfortunately, I know nothing about rust..

(R40)  [ymwang @ ~/Terminus]$ cat salmon_infRep20_out/quant_salmon.sh
for line in $(cat ../Run_GSE112055)
do
        salmon quant -i /home2/ymwang/linqin/RNA-seq/fasta/release99/mouse_rna_salmon_k31_index  -l A -1 ../download_GSE112055/$line".sra_1.fastq" -2 ../download_GSE112055/$line".sra_2.fastq" -p 32 --validateMappings -o quant_out/$line --numGibbsSamples 20 --seqBias --gcBias --dumpEq && echo $line Done!
done
(R40)  [ymwang @ ~/Terminus]$

(R40)  [ymwang @ ~/Terminus]$ terminus group -d salmon_infRep20_out/quant_out/SRR6868519 -o group_out/
------input configuration------
seed : 10
min-spread : 0.1
tolerance : 0.001
dir : salmon_infRep20_out/quant_out/SRR6868519
output folder: group_out//SRR6868519
------------------------------
# targets : 137934
did serialize eq classes : true
# boot : 20
parsing eqfile "salmon_infRep20_out/quant_out/SRR6868519/aux_info/eq_classes.txt.gz"
thread 'main' panicked at 'called `Option::unwrap()` on a `None` value', src/util.rs:1401:33
note: run with `RUST_BACKTRACE=1` environment variable to display a backtrace
(R40)  [ymwang @ ~/Terminus]$

(R40)  [ymwang @ ~/Terminus]$ RUST_BACKTRACE=full terminus group -d salmon_infRep20_out/quant_out/SRR6868519 -o group_out/
------input configuration------
seed : 10
min-spread : 0.1
tolerance : 0.001
dir : salmon_infRep20_out/quant_out/SRR6868519
output folder: group_out//SRR6868519
------------------------------
# targets : 137934
did serialize eq classes : true
# boot : 20
parsing eqfile "salmon_infRep20_out/quant_out/SRR6868519/aux_info/eq_classes.txt.gz"
thread 'main' panicked at 'called `Option::unwrap()` on a `None` value', src/util.rs:1401:33
stack backtrace:
   0:     0x56553760b655 - backtrace::backtrace::libunwind::trace::h14d338b30b3ea0a7
                               at /cargo/registry/src/github.com-1ecc6299db9ec823/backtrace-0.3.46/src/backtrace/libunwind.rs:86
   1:     0x56553760b655 - backtrace::backtrace::trace_unsynchronized::h73ea91d74a3fd67f
                               at /cargo/registry/src/github.com-1ecc6299db9ec823/backtrace-0.3.46/src/backtrace/mod.rs:66
   2:     0x56553760b655 - std::sys_common::backtrace::_print_fmt::hd42948c952866e12
                               at src/libstd/sys_common/backtrace.rs:78
   3:     0x56553760b655 - <std::sys_common::backtrace::_print::DisplayBacktrace as core::fmt::Display>::fmt::ha8f928866ff7571e
                               at src/libstd/sys_common/backtrace.rs:59
   4:     0x565537631dcc - core::fmt::write::he0c1e5f7426d2718
                               at src/libcore/fmt/mod.rs:1076
   5:     0x5655376084e2 - std::io::Write::write_fmt::hf3afc6cfd57d0033
                               at src/libstd/io/mod.rs:1537
   6:     0x56553760dcf0 - std::sys_common::backtrace::_print::hfc0110703f3696fd
                               at src/libstd/sys_common/backtrace.rs:62
   7:     0x56553760dcf0 - std::sys_common::backtrace::print::h3f77c6990ddfaa22
                               at src/libstd/sys_common/backtrace.rs:49
   8:     0x56553760dcf0 - std::panicking::default_hook::{{closure}}::heae49580a8d62d75
                               at src/libstd/panicking.rs:198
   9:     0x56553760da3c - std::panicking::default_hook::hecc34e3f729e213c
                               at src/libstd/panicking.rs:217
  10:     0x56553760e333 - std::panicking::rust_panic_with_hook::he82f5d0644692441
                               at src/libstd/panicking.rs:526
  11:     0x56553760df2b - rust_begin_unwind
                               at src/libstd/panicking.rs:437
  12:     0x56553762fd31 - core::panicking::panic_fmt::h09c929f06bb87c98
                               at src/libcore/panicking.rs:85
  13:     0x56553762fc7d - core::panicking::panic::h7ece43057e5422d4
                               at src/libcore/panicking.rs:50
  14:     0x5655374c6ec5 - terminus::util::parse_eq::ha92665b9854897d2
  15:     0x5655374e9193 - terminus::main::h8313fd0159e1c107
  16:     0x5655374f8b10 - std::rt::lang_start::{{closure}}::hd3e71771a43db155
  17:     0x56553760e7d3 - std::rt::lang_start_internal::{{closure}}::h5d3ea623498f5f43
                               at src/libstd/rt.rs:52
  18:     0x56553760e7d3 - std::panicking::try::do_call::hac65e71be769a440
                               at src/libstd/panicking.rs:348
  19:     0x56553760e7d3 - std::panicking::try::hd4706e264bcf6712
                               at src/libstd/panicking.rs:325
  20:     0x56553760e7d3 - std::panic::catch_unwind::h948a0fb4a8b3ee82
                               at src/libstd/panic.rs:394
  21:     0x56553760e7d3 - std::rt::lang_start_internal::h72cc068ed2d0ac53
                               at src/libstd/rt.rs:51
  22:     0x5655374ec1f2 - main
  23:     0x7f63ded12555 - __libc_start_main
  24:     0x5655374b91a9 - <unknown>
(R40)  [ymwang @ ~/Terminus]$

Best, Ci

[hiraksarkar]

Hi Qin,

Thanks for using Terminus. It seems Terminus panicked while parsing the salmon_infRep20_out/quant_out/SRR6868519/aux_info/eq_classes.txt.gz file. My guess is that Terminus is not getting the format it expects from the equivalence class file. If this is made from the experiment SRR6868519, I can try to reproduce the error when I get a chance. Before that can you please tell me what version of Salmon are you using and which index the salmon is run with.

[Ci-TJ]

@hiraksarkar Thank you very much for your timely response. I use mouse Ensemble release 99 cdna(ftp://ftp.ensembl.org/pub/release-99/fasta/mus_musculus/cdna/Mus_musculus.GRCm38.cdna.all.fa.gz) and ncrna(ftp://ftp.ensembl.org/pub/release-99/fasta/mus_musculus/ncrna/Mus_musculus.GRCm38.ncrna.fa.gz) to build index by salmon(v 1.2.1), which built index with k=31 and other parameters by default. I will send my index to your email. By the way, I have update my version of salmon to v 1.3.0 in my current conda env. Does terminus call salmon during runtime?

[hiraksarkar]

Hi @Ci-TJ ,

It does not call Salmon internally and salmon(v 1.2.1) should ideally work. I would definitely check SRR6868519 when I get a chance and get back to you if I can reproduce the result.

[hiraksarkar]

Hi @Ci-TJ ,

Can you please re-run the salmon with the following command in your script

salmon quant -i /home2/ymwang/linqin/RNA-seq/fasta/release99/mouse_rna_salmon_k31_index  -l A -1 ../download_GSE112055/$line".sra_1.fastq" -2 ../download_GSE112055/$line".sra_2.fastq" -p 32 --validateMappings -o quant_out/$line --numGibbsSamples 20 --seqBias --gcBias -d

Notice the use of -d instead --dumpEq, which does not provide rich equivalence classes (equivalence classes with weights), a requirement for terminus.

[Ci-TJ]

Hi @hiraksarkar It's no problem in group, but I got trouble in collapse.

(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$ cat salmon_quant_terminus.sh
### quant for terminus
pwd
salmon -v
terminus --version
date
for line in $(head -n 4 ../Run_GSE112055)
do
        salmon quant -i /home2/ymwang/linqin/RNA-seq/fasta/release99/mouse_rna_salmon_k31_index  -l A \
                -1 ../download_GSE112055/$line".sra_1.fastq" -2 ../download_GSE112055/$line".sra_2.fastq" -p 32 --validateMappings -o quant_out/$line --numGibbsSamples 20 --seqBias --gcBias -d && echo $line Done!
        terminus group -d quant_out/$line -o group_out && echo group $line OK!
done

date(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$ ll group_out/SRR6868519/
total 159240
drwxrwxr-x 2 ymwang ymwang      118 Nov 29 00:13 ./
drwxrwxr-x 6 ymwang ymwang       94 Nov 29 00:38 ../
-rw-rw-r-- 1 ymwang ymwang   438614 Nov 29 00:13 collapsed.log
-rw-rw-r-- 1 ymwang ymwang 97092240 Nov 29 00:13 delta.log
-rw-rw-r-- 1 ymwang ymwang 62018524 Nov 29 00:11 die_roll.log
-rw-rw-r-- 1 ymwang ymwang    72122 Nov 29 00:13 groups.txt
-rw-rw-r-- 1 ymwang ymwang  3427455 Nov 29 00:11 infrv.log
(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$

(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$ terminus collapse -h
terminus-collapse
analyze a collection of per-sample groups, and produce a consensus grouping.

USAGE:
    terminus collapse [OPTIONS] --dirs <dirs>... --out <out>

FLAGS:
    -h, --help       Prints help information
    -V, --version    Prints version information

OPTIONS:
    -c, --consensus-thresh <consensus-thresh>    threshold for edge consensus [default: 0.5]
    -d, --dirs <dirs>...                         direcotories to read the group files from
    -o, --out <out>                              prefix where output would be written
    -t, --threads <threads>
            number of threads to use when writing down the collapsed quantifications [default: 8]

(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$ terminus collapse -d group_out/SRR6868519/ group_out/SRR6868520/ group_out/SRR6868521/ group_out/SRR6868522/ -o collapse_out
experiment name
thread 'main' panicked at 'called `Result::unwrap()` on an `Err` value: Os { code: 2, kind: NotFound, message: "No such file or directory" }', src/util.rs:267:38
note: run with `RUST_BACKTRACE=1` environment variable to display a backtrace
(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$ terminus collapse -d $PWD/quant_out/SRR6868519/ $PWD/quant_out/SRR6868520/ $PWD/quant_out/SRR6868521/ $PWD/quant_out/SRR6868522/ -o collapse_out
experiment name
# targets : 137934
did serialize eq classes : true
# boot : 20
thread 'main' panicked at 'called `Result::unwrap()` on an `Err` value: Os { code: 2, kind: NotFound, message: "No such file or directory" }', src/main.rs:224:63
note: run with `RUST_BACKTRACE=1` environment variable to display a backtrace
(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$ RUST_BACKTRACE=1 terminus collapse -d $PWD/quant_out/SRR6868519/ $PWD/quant_out/SRR6868520/ $PWD/quant_out/SRR6868521/ $PWD/quant_out/SRR6868522/ -o collapse_out
experiment name
# targets : 137934
did serialize eq classes : true
# boot : 20
thread 'main' panicked at 'called `Result::unwrap()` on an `Err` value: Os { code: 2, kind: NotFound, message: "No such file or directory" }', src/main.rs:224:63
stack backtrace:
   0: backtrace::backtrace::libunwind::trace
             at /cargo/registry/src/github.com-1ecc6299db9ec823/backtrace-0.3.46/src/backtrace/libunwind.rs:86
   1: backtrace::backtrace::trace_unsynchronized
             at /cargo/registry/src/github.com-1ecc6299db9ec823/backtrace-0.3.46/src/backtrace/mod.rs:66
   2: std::sys_common::backtrace::_print_fmt
             at src/libstd/sys_common/backtrace.rs:78
   3: <std::sys_common::backtrace::_print::DisplayBacktrace as core::fmt::Display>::fmt
             at src/libstd/sys_common/backtrace.rs:59
   4: core::fmt::write
             at src/libcore/fmt/mod.rs:1076
   5: std::io::Write::write_fmt
             at src/libstd/io/mod.rs:1537
   6: std::sys_common::backtrace::_print
             at src/libstd/sys_common/backtrace.rs:62
   7: std::sys_common::backtrace::print
             at src/libstd/sys_common/backtrace.rs:49
   8: std::panicking::default_hook::{{closure}}
             at src/libstd/panicking.rs:198
   9: std::panicking::default_hook
             at src/libstd/panicking.rs:217
  10: std::panicking::rust_panic_with_hook
             at src/libstd/panicking.rs:526
  11: rust_begin_unwind
             at src/libstd/panicking.rs:437
  12: core::panicking::panic_fmt
             at src/libcore/panicking.rs:85
  13: core::option::expect_none_failed
             at src/libcore/option.rs:1269
  14: terminus::main
  15: std::rt::lang_start::{{closure}}
  16: std::rt::lang_start_internal::{{closure}}
             at src/libstd/rt.rs:52
  17: std::panicking::try::do_call
             at src/libstd/panicking.rs:348
  18: std::panicking::try
             at src/libstd/panicking.rs:325
  19: std::panic::catch_unwind
             at src/libstd/panic.rs:394
  20: std::rt::lang_start_internal
             at src/libstd/rt.rs:51
  21: main
  22: __libc_start_main
  23: <unknown>
note: Some details are omitted, run with `RUST_BACKTRACE=full` for a verbose backtrace.
(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus]$

Do I misunderstand with input of collapse?

[hiraksarkar]

Hi @Ci-TJ ,

There is a directory dependency between group and collapse, So your group command should write the output in the same location where you want to write your collapse output in,

For example if you have three experiments, SRR1852860-81

terminus group -m 0.05 -d quant/SRR1852860/ -o term_out/SRR1852860
terminus group -m 0.05 -d quant/SRR1852861/ -o term_out/SRR1852861
terminus group -m 0.05 -d quant/SRR1852881/ -o term_out/SRR1852881

Your collapse command will be as follows,

terminus collapse -d quant/SRR1852860 quant/SRR1852861 quant/SRR1852881 -c 1.0 -o term_out

In your case, you can do the following,

terminus collapse -d group_out/SRR6868519/ group_out/SRR6868520/ group_out/SRR6868521/ group_out/SRR6868522/ -o group_out

[Ci-TJ]

@hiraksarkar Thank you for you help during this time! I have reproduced your codes, and I have a question if I can feed directly the outpout of collapse to fishpond for differential expression analysis. In any case, I will try later.

Best

[hiraksarkar]

Hi @Ci-TJ

Glad to hear. You can use the code directly in DE-seq2 by following the tutorial, https://combine-lab.github.io/terminus-tutorial/2020/running-terminus/

[Ci-TJ]

Yeah, I know how to use the DESeq2 for DE analysis, but fishpond is special and easy-to-use for salmon; If outputs of terminus can be fed directly to fishpond, it's very wonderful!

[hiraksarkar]

Hi @Ci-TJ

I just had a brief conversation with Mike Love (who wrote fishpond), Can you please put an issue there with how you wanna try fishpond. He might add some added code to make that work. Feel free to tag me too.

Thanks

[Ci-TJ]

Hi @hiraksarkar @mikelove I have tried to import the outputs of teminus directly by tximeta, but failed.

(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus/fishpond]$ cat ../salmon-terminus.sh
### quant and terminus
echo ==================================================================================
echo ==================================================================================
pwd
salmon -v
terminus --version
date
for line in $(cat ../Run_GSE112055)
do
        salmon quant -i /home2/ymwang/linqin/RNA-seq/fasta/release99/mouse_rna_salmon_k31_index  -l A \
                -1 ../download_GSE112055/$line".sra_1.fastq" -2 ../download_GSE112055/$line".sra_2.fastq" -p 32 --validateMappings -o quant/$line --numGibbsSamples 20 --seqBias --gcBias -d && echo $line Done!
        terminus group -m 0.5 -d quant/$line -o term_out && echo group $line OK!
done

# for collapse
ls quant
terminus collapse -d quant/SRR6868519 quant/SRR6868520 quant/SRR6868521 quant/SRR6868522 quant/SRR6868523 quant/SRR6868524 quant/SRR6868525 quant/SRR6868526 quant/SRR6868527 quant/SRR6868528 -c 1.0 -o term_out
date
(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus/fishpond]$ ll ../term_out/SRR6868519/
total 369552
drwxrwxr-x  3 ymwang ymwang      4096 Nov 30 10:28 ./
drwxrwxr-x 12 ymwang ymwang      4096 Dec  2 19:34 ../
drwxrwxr-x  3 ymwang ymwang        55 Nov 30 10:28 aux_info/
-rw-rw-r--  1 ymwang ymwang     50441 Dec  2 19:35 clusters.txt
-rw-rw-r--  1 ymwang ymwang       465 Dec  2 19:35 cmd_info.json
-rw-rw-r--  1 ymwang ymwang    423733 Dec  2 18:17 collapsed.log
-rw-rw-r--  1 ymwang ymwang  94214845 Dec  2 18:17 delta.log
-rw-rw-r--  1 ymwang ymwang 274633069 Dec  2 18:15 die_roll.log
-rw-rw-r--  1 ymwang ymwang     69588 Dec  2 18:17 groups.txt
-rw-rw-r--  1 ymwang ymwang   3424114 Dec  2 18:15 infrv.log
-rw-rw-r--  1 ymwang ymwang   5578342 Dec  2 19:35 quant.sf
-rw-rw-r--  1 ymwang ymwang        68 Dec  2 19:35 terminus_info.json
(R40)  [ymwang @ ~/linqin/RNA-seq/Analysis/GSE112055/Terminus/fishpond]$

> library(tximeta)
> library(fishpond)
> suppressPackageStartupMessages(library(SummarizedExperiment))
> dir<- "/home2/ymwang/linqin/RNA-seq/Analysis/GSE112055"
> coldata <- read.table("coldata.txt", sep="\t", header=T)
> head(coldata)
       names    BioSample drug_treatment Experiment GEO_Accession Sample.Name
1 SRR6868519 SAMN08741761           None SRX3822981    GSM3056373  GSM3056373
2 SRR6868520 SAMN08741758           None SRX3822982    GSM3056374  GSM3056374
3 SRR6868521 SAMN08741757           None SRX3822983    GSM3056375  GSM3056375
4 SRR6868522 SAMN08741754           None SRX3822984    GSM3056376  GSM3056376
5 SRR6868523 SAMN08741749           None SRX3822985    GSM3056377  GSM3056377
6 SRR6868524 SAMN08741742           None SRX3822986    GSM3056378  GSM3056378
  surgery  X
1    Sham NA
2    Sham NA
3    Sham NA
4    Sham NA
5    Sham NA
6     TAC NA
> coldata$files <- file.path(dir, "Terminus", "term_out",coldata$names, "quant.sf")
> file.exists(coldata$files)
 [1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
> head(coldata)
       names    BioSample drug_treatment Experiment GEO_Accession Sample.Name
1 SRR6868519 SAMN08741761           None SRX3822981    GSM3056373  GSM3056373
2 SRR6868520 SAMN08741758           None SRX3822982    GSM3056374  GSM3056374
3 SRR6868521 SAMN08741757           None SRX3822983    GSM3056375  GSM3056375
4 SRR6868522 SAMN08741754           None SRX3822984    GSM3056376  GSM3056376
5 SRR6868523 SAMN08741749           None SRX3822985    GSM3056377  GSM3056377
6 SRR6868524 SAMN08741742           None SRX3822986    GSM3056378  GSM3056378
  surgery  X
1    Sham NA
2    Sham NA
3    Sham NA
4    Sham NA
5    Sham NA
6     TAC NA
                                                                                  files
1 /home2/ymwang/linqin/RNA-seq/Analysis/GSE112055/Terminus/term_out/SRR6868519/quant.sf
2 /home2/ymwang/linqin/RNA-seq/Analysis/GSE112055/Terminus/term_out/SRR6868520/quant.sf
3 /home2/ymwang/linqin/RNA-seq/Analysis/GSE112055/Terminus/term_out/SRR6868521/quant.sf
4 /home2/ymwang/linqin/RNA-seq/Analysis/GSE112055/Terminus/term_out/SRR6868522/quant.sf
5 /home2/ymwang/linqin/RNA-seq/Analysis/GSE112055/Terminus/term_out/SRR6868523/quant.sf
6 /home2/ymwang/linqin/RNA-seq/Analysis/GSE112055/Terminus/term_out/SRR6868524/quant.sf
> se <- tximeta(coldata)
importing quantifications
reading in files with read_tsv
1 2 3 4 5 6 7 8 9 10
Error in if (!is.na(m)) { : argument is of length zero

By the way, I can import the outputs of salmon's quant to fishpond by tximeta. What's the solution?

[mikelove]

Can you try with tximport instead? Or with skipMeta=TRUE? The crux is that tximeta typically works by attaching the transcript ranges to the quantification data, but after Terminus, the rows are no longer transcripts but transcript groups. I'd like to develop a solution for Terminus output but this doesn't exist yet in tximeta.

[Ci-TJ]

@mikelove It's working when I import outputs of salmon by tximeta with skipMeta=TRUE. I didn't try tximport, because I thought it would be work too. For now, there are some problems for processing DE annalysis for gene-level, but it's doesn't matter. I will look forward to next update of tximeta. Thanks