Detected no BSJ using psirc_v1.0.pl

Dear Dr. Christina Huan Shi,

Thank you for this great software. This software is very helpful and incredibly fast. But when I run it using my own data, I got a result unexpectedly.

I ran psirc according to the pipeline you recommend here. (1) I created the custom transcriptome fasta file using create_custom_transcriptome_fa.pl which accepts the genome fasta file hg38.fa and the gtf file gencode.v34.annotation.gtf as input. (2) I created the index of the custome transcriptome generated in (1) using psirc_v1.0.pl. I'm quite sure of the success of the creation of the index after I checked the file carefully. (3) I went ahead with producing both BSJ detection and FLI inference outputs in a single run using my own data as you recommend:

My command is:

perl psirc_v1.0.pl -t 10 -f -o output gencode.v34.annotation.psirc.custom_transcriptome.fa kallisto fastq1 fastq2

where fastq1 and fastq2 is first-pair and second-pair fastq file of a paired-end total RNA-seq respectively. And the read length of the fastq uis 150-bp.

The script ran normally and ended in several minutes.But when I checked the output folder, I found something unreasonable. I found that the candidate_circ_supporting_reads.sam, candidate_circ_junctions.fa and candidate_circ_junctions.bed are all empty. and when I check the log file generated during the process. The content is:

Mapping reads to all_possible bsj targets... 2021年 11月 28日星期日 01:46:53 CST Finished mapping. 2021年 11月 28日星期日 01:47:44 CST Mapping reads to FirstandLastExon entities... 2021年 11月 28日星期日 01:47:44 CST Finished mapping. 2021年 11月 28日星期日 01:48:22 CST Extracting possible bsj reads... 2021年 11月 28日星期日 01:48:22 CST Finished extracting. 2021年 11月 28日星期日 01:49:40 CST Extracting potential backsplice transcripts... 2021年 11月 28日星期日 01:49:40 CST Finished extracting. 2021年 11月 28日星期日 01:49:42 CST Final mapping... 2021年 11月 28日星期日 01:49:42 CST Final round confirmation of backsplice segments... Number of backspliced unique reads: 0

Number of candidate circular RNA junctions: 0 Finished mapping. 2021年 11月 28日星期日 01:49:42 CST

Full length isoform inference... 2021年 11月 28日星期日 01:49:42 CST Mapping reads to fsj targets... 2021年 11月 28日星期日 01:52:12 CST Finished mapping. 2021年 11月 28日星期日 01:52:43 CST Extracting potential full-length circRNA isoforms... 2021年 11月 28日星期日 01:52:43 CST Finished extracting. 2021年 11月 28日星期日 01:52:46 CST Extracting potential fsj reads... 2021年 11月 28日星期日 01:52:46 CST Finished extracting. 2021年 11月 28日星期日 01:54:05 CST Final mapping... 2021年 11月 28日星期日 01:54:06 CST Final round confirmation of fsj segments... Total number of genomic loci: 223106 linear (input annotation): 223106 circular (back-splicing junction): 0

Number of full-length isoforms: 228082 linear (input annotation): 228048 circular (back-splicing junction): 0 linear alternative (exon skipping): 34 circular alternative (exon skipping): 0 Finished full length isoform inference. 2021年 11月 28日星期日 01:54:19 CST

So it seems that psirc did not detect any potential back-spliced junciton reads. It's unexpected for me considering that the dataset I used here is a total RNA-seq data form HEK293T and I had detected thousands of circular RNAs using other softwares such as CIRI2, find_circ, etc. So I think that maybe there is something wrong with my pipeline or your software. Could you please help me check the pipeline you provide here or provide the pipeline in detail you used in the Genome Research paper? I' ll appreciate your help vey much.

Yours sincerely,

sui liang.

Hi Sui Liang, thanks for your interest in the tool. Have you also made sure that "kallisto" is the forked kallisto that can be downloaded as an executable under the Requirements section? It's required to to provide the path to the forked kallisto there since other versions of kallisto will not work for this step.

The other thing I recommend trying is to use the custom_transcriptome_fa we provide (gencode.v29.annotation.custom_transcriptome.fa) also under Requirements as a test to see if it works first. If it works, then probably the generation of another one using create_custom_transcriptome_fa.pl has some problems for your use case that we can also debug together.

Hi Dr. Christina Huan Shi,

Thanks for your timely reply and recommendation. I have done the things according to your suggestion.

(1) I'm sure I had used the forked kallisto you provided here. (2) I found that the custom transcriptome fasta file you provided was in Google Drive. Unfortunately, I cannot visit it because of the constraint of Chinese network. So l used gencode.v29.annotation.gtf to create the file for myself and then ran the pipeline again. But unfortunately, it returned no BSJ again. When I checked the log file carefully, I found that it began with this:

[quant] fragment length distribution is truncated gaussian with mean = 30, sd = 20 [index] k-mer length: 31 [index] number of targets: 9,372,965 [index] number of k-mers: 73,536,645 [index] number of equivalence classes: 11,195,812 [quant] running in single-end mode [quant] will process file 1: E0_1_1.clean.fq.gz [quant] will process file 2: E0_1_2.clean.fq.gz [quant] finding pseudoalignments for the reads ... [quant] fragment length distribution will be estimated from the data [index] k-mer length: 21 [index] number of targets: 206,694 [index] number of k-mers: 101,367,781 [index] number of equivalence classes: 929,051 [quant] running in paired-end mode [quant] will process pair 1: E0_1_1.clean.fq.gz E0_1_2.clean.fq.gz [quant] finding pseudoalignments for the reads ... [build] loading fasta file output/mapping/potential_backsplice_transcripts.fa [build] k-mer length: 21 [build] counting k-mers ... done. [build] building target de Bruijn graph ... done [build] creating equivalence classes ... done [build] target de Bruijn graph has 0 contigs and contains 0 k-mers

[quant] fragment length distribution will be estimated from the data [index] k-mer length: 21 [index] number of targets: 0 [index] number of k-mers: 0 [index] number of equivalence classes: 0 [quant] running in paired-end mode [quant] will process pair 1: output/mapping/possible_bsj_reads_1.fastq.gz output/mapping/possible_bsj_reads_2.fastq.gz [quant] finding pseudoalignments for the reads ...sh: 行 1: 14748 段错误 (吐核) biosoft/psirc-master/forked_kallisto/Linux/kallisto pseudo -i output/mapping/potential_backsplice_transcripts.fa.index -o output/mapping -t 1 --pseudobam output/mapping/possible_bsj_reads_1.fastq.gz output/mapping/possible_bsj_reads_2.fastq.gz > output/mapping/temp_final_mapping.sam

As you can see, it seems that the piepline encountered an exception when creating the index for potential_backsplice_transcripts.fa. On account of this, I checked output/mapping/potential_backsplice_transcripts.fa and I found that this file was empty. So I think that there's something wrong at the beginning.

I'm sure that I had done the things according to the guide. (1)I generated the custom fasta file with this command:

perl create_custom_transcriptome_fa.pl hg38.fa gencode.v29.annotation.gtf gencode.v29.annotation.psirc.custom_transcriptome.fa

The gencode.v29.annotation.psirc.custom_transcriptome.fa looks like this:

chr11:133896437-133901601_ENST00000624667.1;MIR4697HG_Exon_Lengths_5164_Offsets0- TTTTTTAGCAAATGTTTATTTATATCAAAATACAAAACATTTCTGAAGCAGAGTAATGTTACATGAGGAGCAATTAAATACTAAACGTCTTTTCATCAGTTTAACTTGCAACACTCCCGGGGTACCTGCACTGGCTGCACATCTCATCATTTGATGTGTGCCTTCTTTTTTCTCATTTGCTTATGCACGTGTTTGCTCATTTTGATTTGTAATTCAGGATATGAACATGCAGTGTTACTGGGGATGAGTTGGTTAGTACTTAGCAAGCCTGGAAATACCAGCGAGTGGAGCCTGGCGTGGTAGCCTGAGCTGAGGCAGGCTGCAGGTTCTCTTCCTGACTGAGTCTGGGGTGTCACTCCATTTTTGACTGCCAAGTCAAAGGACAAAGGGATCACCATCATCAGCAGCCAGGGCAGGCGTCTATTGCTTAGGGCCGTAGAGCAGGCGTGGCTTTGGGGTCATCTCCAGCGGGGTCTTCTCAGTGTTTGGGAAGAAGGGGCAGGGCAGTCTATAGGCCCAGGCCCAGCAGCAGGATGCCCAGGGTCCCTGCAGGAAGAACCCTCTTCCCCAGGAAAGCAAGGAAGGGTTCGGGAGGAGACAGTTCCTCCTACTCTTGGAAGTCTGTGATTTGGTGGCTGGGGGTGCCTGCCCATTGGAAGGGCCCCTTATAAATGCTTTGAGTTTGGAGATTTTGAGGGAAAAAAAGCACCTGATTGAGAGTGTAGCTGTTTCCAATACACCTCCCTTCCCTGACGGAGAAGGGAAGGAGGCAGAAAACAGGGGAGAGAGCATCCCCCCTTCATTCCCACACCGCCCCACTCCCAGAGCCAACCTGGGCCTCCCTTCAAGCTCGTACTTAGTACATTCACCATGACACTGTGCACCTGTGAGCTCTCTCCCGCTCTCACTCTCTTGCGTGCACACACGCACACGCACACACACGCACAGGTATGCACACATATATATGCAGGTGCGCGCACACACACACATCTGGTTTCCTTGCAGTCTGCTCAGGCAGCAGAAGGGTGTCCCATCCTCACCGTCATCTTTTACTTCCCAGGGTGCCCTTGGCCCACAGGTCCCCAAGACCCAGCCCCAGGCTTGGCACGCGCTGGCATTCCCAGGCTTTTGAGAAGAGGGTGATAGAGCAGGGAGCTCCTCACAGACAGGTTATACAGAGTGTAGAATATGTTCATATAAGGCTTTGGTACAGCACCGATTTCATGTTCTTATAGTTTGATTTCTAAGTCATCTTTTAGAAGAAAAAAAAGCTTACTGAAAAAATAGTGGTTTTATTTACTTTTGAGTTACATACCTGAAGTTCAAAAAGTAAGCCTCTAGTTGCCATTCAAGTTCACACTCAAAGGTGTTCTGTGTGCAGCTGTACAATTTGTTGGTTTGTCTTTAATGTTGAACAAAAGTCAGCCAACCCATACCATAAAGTCAGCTGCCCAAACTCACTTTAACCAGTCCATACATACATACAGAAAAGAAAGAGAGAGAGAGGGAGAACACAAAAGCCGCCCCTAAGTCACCCACCCCAAACCCCTAGGGAAAAATCAGAAAGAAGACAAAATACAGCAAGATATCAGGAGCCAGTGGGAGGAAGGAGATTCAGCCCAACTGGCCTCTTCGAGAACAGCACCATGAAGCACCCTAGCGCAGTGATGTTGTAATACTCAACATTCAAGAAATGGAAACGAGGTTGGAACGGTCTTCACAGGAAGACCTGGCATGGATCGGGGGTCGCCCCCAAAGAAAGTGGAAGAAAGAAAGAGTTCGAAATGAGAGCAAGAAATCTCATGACGAAACTGACAGTCATATCCAGAAAAGAAGAACTACAGCAAGAGGCTGCCGCGCACCGAGAGGTACAGAGTTTGTAAGGGGTCTGGGGGAGGTAGCCCCTCCCCACCCACACTGCCTGCTCACCCCCACCCTCTGTGTGTCCTCAGACCACCAGGTGAAATGGAGGAAAGAAGCTCATCTAAAACTTGCCCCCAGGTGTGCGATGGGAGAATTCTATCCTGCATGATCAAAGTCCCTCCTGGGGAAGAGAAAAACACTGAAGACCAAGGGGCAGGAGTCACTGACATGAAGCGGGATGTGGTCCCTTCACGTCAGTGACTGCGCCCCCTTCTGGGCCCTCTATCCCCAGCCCCACACCTGGCAGTTAGGAAATTAAATCTGAATGGGAGGTCAAGACCTTCTGACACTGATACAGACGACTTAGCAAGTGGAGCGTTAAACTGCTCTTCTCCCCTAGCCAACAATTCCCCTTTCTAACACCTCAGTCTAAAGTTCCAGAATCAAGACCATCCCATCAAGTACCTTCCTCCCAGTCCCTGGACTCTGAGACTCCACCCTCCAATGCCACAGGCAACCCCCTGTGGACAGGAAGTGTGTGTGCAGGCTTGCCTCTTCCCCCACCTCCTACTTTCAGACCGCCCAAAGTGGATCACCATGAACATCTACCGCCAAGAAGAGTGGAAGCGGGAAGCAGGCAGCAAAAATCCACGACAGAGCCTTTTCCCCAAGCTGCTGGTTGGTGACAGTGGGGAAGAGGGACAGGGCCCACACTGATCCATGGGAGAGTGATTCTCACACATCTCCCTATTCCCGACTCTTGCTCTATATAGTAGACAGAGGTAGCAAGACAGAAACAACTCCTCAGATACCTCCACCTTCTACCAAAGGGAAAAGCACCAAAATTCAATGTCCATTCTGCTGTGGGGACCAAGGAACGTGTCCCAAGCATGGGGAAGAGCTCAGGCTCCTGGCAGTGGCTGTGGGCCGTAAAAGCTCCATCAGAGCACACTCATCCACCTGTCGCCCCAACTGATGCCACAAGCGGTTCCCAGGCCTCCTCCTTACTCCACATAAAGCTGATCTCAATGACCTTTGCTCCAAGTGCCTTAAGGGCTGGCTCTTCAGAGAAGCTGGAGGTCCCCTCTGACACCCAACCTTACCTCATCTTGCTGAGCAGTGGAGCAGAAAGGTCACAGGTAGGCACTAACATCCCCTCCACACTGTACAATTTCAAGGTAGGGGCTATGGCCCCATCTTCCCTTTCCCTCCCCAAAATGAAGAGTCCTGTTGGAGGCTGAGGAATAGAATGGTGACATGTGGGCCAGTGGCTCTCCCAGACCTTGATGCTGCTGAGGACCTAATCTTGGGCTGGCCCTGGTGGGAGGGCAAGCCCTAGGACATGCCCGAGGTGTTGGAGCAGTTTACTCTGCTGGTTGCATGTGGCCAGAAGTCGGGCAGAGTCCACAGAAAGAGAGGGGATGGTGGGTCTAGAAGCCAACAGAACAGTAATGGGAGCCTCCCTTGTGCAGAGACCGTGGTGGTCTGGGAAGGCTTCAAGGTAGGAAGGGAACAGAATTTGCACCTCTTCTTCTTGCCCAATCCCCCTGAAAAATCTTACAAGAAACACTCAGCTGCAGGAGCCTGCAAGAAAGATCATTCAAGGCAAGGATGGTGCCACTGACAGTGGCACCAGCACTGGATCCTTCCAGCTGCATGGACAGAGCCCATTTCCCATCAGCGTCCTCGGAAAGTGGGGAGTGTTCTCAGCAGTTAATTTGAGGACCCAAGGACAGTGCCTGACTGAATGGCAGGGCCTCATAGGTCCATGTAAGGATCAAGAGTTAGACTCAAGCTTTACCCCAACCTACTCTTCCCATAATACCCCAAAACTGTCAACCAAGCCCTTCTCACGCTGAAGGTATCTGCTTTCCTCTAAGGACCAAACTCTATAGACAAGAGCTCTTAGGGAGTGCCATGTCTTGGTTAGGAAAGGGGTTAGTACTGCCCTTTTCTGAGTGATGAGCAGGAATAAGGAGCCGAAAGGTTGCAGTCCTGAGTCTCTCCTCACTGCCTGCACCATCTGGCCCAGCACCAGCTTCAAATACATCACTCCGAGCTCCGGGCAAGTCAGGAAGGAGCTAGCACCAGGAGACGCTTGCTGAGGCTGCAAGGCAGAGCCCTTGCATATGTGTGAGGTCTGGAATTTGCTCAGAGAGGTATTCTTCAGGGCACACTGCTTCCTGCTCAGATACAAGTGAACTCCTGCTCCCCAGAATCTCCCATTGTGGGTCCAGTTCTGCCAATACATTGTTCAGTTTTGTAAGTCCCCGGCTGGAGGCCAGAAGGAAGGTTCCACTAAGACCCCCTCTCCCAGGCCGGCCCACAGTTCATCTCTCCCTCCTCATCCTCCACTCAGTGTCCCAGGATTGGAGCATCCTATTTGCAATGGCACTTTATTTCCAATTAAAAATAACTGAAAAAAACATAGATGACATATTTATACTACATACCACATTCACACTGGCTCTAAGAATCTGCAGCAACACTAAACAGGCCATGTGTTCTCCAAGACCAAGCCCAGGCAGCCAGCCTCACAAACTCTCCCATTGCCATGCCCTCTATGAGCGGTCTGAGCTGTCTGAGCTACAGTGCTACAAACCAAGACAGGTGTCACCGTAGGTCCCTAGGTAGTCTCAACCACCCCCTTTCTAATGCTGTGAAGCCAGGTTTTCTCTGAGGAACTGGTTACCTTTCCACAGCCACGTGGGGATGGAGTCACAGTTTTCATCTCCTCAGCCTTGCCCTGTTAAGCCCCTCTGCAAAGAAGTTCAAGTCAACATCCCTCCCAAGTGGCAAGAGACACATTGGTCCTGGTTCTTTAGGGCTATCAGATCCTGCAGCTCCACACAACCTTCAGCTCACTTGTCCTTCCTGCAGGTGGGTTATTTGTTTTGTTATTTTACAAACTTTTCATATATACACATTTCCATCAGAAAGACCCAAGCAACTCTGAACAGAGAGAATACCAAAGGAAGAGCAGGGAAGGAGGTGGCGTCAAGGCATGAGCTAGTATCCCAGACTGCCTGGGAAGGTAGCACTGCTCAAAACATCTCTAAGCACAGGTACAAAAATAAAGCAAACTATGACTTCATATAGATATAGAGATATATAGACTTTATCTAGGTGTTCTTTATATTTATATATGTGTGCAGAGGGCACGGGCCCCTGCCCATCTTGCCCGGCACTCATCTCTTGCCCTCCCGCTCCCCACACCCGAGTGCCACTCGCCCGAAGCTCCCAGTCTCCACGATTGCATCTGATCCTTGCCTCCCACATACATTCCCCTCTCTCCTTAAAA chr4:36496692-36641900_ENST00000505298.5;LINC02505_Exon_Lengths_360,62,74,132,109,51_Offsets0,1734,85964,87972,118725,145157- CCATTTAGGCCTCTTTATCCAGGGACTACAAAATAGTAGACTGTTAACTTTTTATTTCATAATTGTATTGGCTGAGCTAAACAGGTTAAATTCAAGACAGCTTAACCCCAAGTTGTCAGCTGGTTATTTGAACAAGTATTCCAGGGCTTTAGTTGAATTCAGGATGAAGTTTCCTCACATCACTTCACTTTTCATCTCTTGCCACAATATCCTAACCTCAGCTTCTGATGTGGTGGATAAGATGACCCCTCCACTCTCTCCAGTCACAATTGCCATGCCCCCACCTGGTGTCTGGAGCAGAAACTCAGGGCATGGGAATCATCGCATTTCCAGCTTTCCACAAATTGAAACTAGTATTGACTTCTTTGTTGTTGTAGTGTCTAGGGTGGTGATAATATGTAGAAGATGTTCAATCAATGCTTCATGATTGCAGGACTAAAAGTTGTTCTGGTGCGTTGGTGAGTCAGGAGTAGAGAAGTTTTTATATTCTATTTTTCATTTTGACCTTATCTCCTTTGCCACAGAAGTTTGAACGTATTCTTCCTGCTCAAGAAAATGCTGTTTCCCCAGAGAATTTCATGTTTGGCTCCTTCTCATCATTCATTCAGGCGTTGGAAAAATGTCACCTAGTTCCACATGGCTGGGGAGGCCTCAGGAAACTTACAATCATGGTGGAAGGTGAAGGAGAAGCAAGCACCTTCTTCACAAAGTGGCAGGATAGAGGAGAGAAGGAGAACCTGGGCGTGATGGCGGGCGCCTGTAATCCCAGCTGCTCCGGAGGCTGAGGC

(2) I generated the index using this command:

perl psirc_v1.0.pl -i gencode.v29.annotation.psirc.custom_transcriptome.fa ~/biosoft/psirc/forked_kallisto/Linux/kallisto This generated 4 files: gencode.v29.annotation.psirc.custom_transcriptome.FirstandLastExons_entities.fa.index gencode.v29.annotation.psirc.custom_transcriptome.all_possible_bsj_targets.fa.index gencode.v29.annotation.psirc.custom_transcriptome.all_possible_bsj_targets.fa gencode.v29.annotation.psirc.custom_transcriptome.FirstandLastExons_entities.fa

and their size looked normally.

(3) Lastly, I produced BSJ detection output:

perl psirc_v1.0.pl -t 10 -o output gencode.v29.annotation.psirc.custom_transcriptome.fa ~/biosoft/psirc/forked_kallisto/Linux/kallisto E0_1_1.clean.fq.gz E0_1_2.clean.fq.gz

Thank you so much for your kindness.

Yours sincerely,

Sui Liang.

Thanks for repeatedly trying to get it to run, and sorry that this error occurs. Indeed it's weird because I've never seen this error before. Based on the output log, it looks like the first two pseudoalignments did not complete, and so it's natural that the intermediate fasta file is empty. How big are your fq.gz files? Would you mind sending them to me through a link so I can try to get it to run as well? If the files are too big, you can subset them using 'seqtk sample' or similar before sending.

Since there are issues in your location to access Google Drive, here is an alternate OneDrive download link for custom_transcriptome_fa: https://1drv.ms/u/s!AmfpM5D0fOvDgX4nzXK1gsDCpW9u?e=pjWuLF

Hi ,

Thanks again for your reply. My fq.gz is about 2.4G and I think it's a little big. So I sampled 100, 000 reads from the file using the command:

seqtk sample -s 100 E0_1_1.clean.fq.gz 100000 | gzip -nc >test_1.fastq.gz seqtk sample -s 100 E0_1_2.clean.fq.gz 100000 | gzip -nc >test_2.fastq.gz [Uploading test_1.fastq.gz…]() [Uploading test_1.fastq.gz…]()

And I' ve attatched these two files here. I really appreciate your helping me solve this problem.

Yours sincerely,

Sui Liang.

test_1.fastq.gz test_2.fastq.gz

I haven't tried your reads yet but I think I was able to reproduce your error with my other input data. Basically, the problem is that the pseudoalignment steps quit very early and don't run at all. The forked kallisto static executable we provided has this issue (even though previously there wasn't this issue in the couple of Linux systems we tested). We will hopefully re-upload a more usable static executable to address this issue soon. Meanwhile, compiling from source worked for me. Download https://github.com/Christina-hshi/kallisto-b to your system, then build it: cd kallisto-b-master mkdir build cd build cmake -DCMAKE_INSTALL_PREFIX:PATH=$HOME .. make make install

The executable in ./src/kallisto should then work. Let me know how it goes

Dear Dr. Ken Hung-On Yu,

Hi. Forgive me for the delay of replying you because of something wrong with the server in my school. Thank you very much for your help. I have run the piepeline with kallisto, version 0.43.1 following your suggestion and my command is:

perl psirc_v1.0.pl -t 10 -o output $fa $kallisto $fastq1 $fastq2

Unfortunately I got this error:

[quant] fragment length distribution is truncated gaussian with mean = 30, sd = 20 Error: pseudobam is not compatible with running on many threads.

According to the message in this error file, I decided to run the piepline using only one thread, namely -t 1. As a result, the task completed normally and returned thousands of candidate circular RNA junctions, just asI expected. Maybe there's still something wrong with kallisto or otherwhere but I don't think it's a severe problem given that the tool is really fast even if executed in single threaded mode. I really appreciate your help very much. Thank you once again.

Best regards,

Sui Liang.

Hi Sui Liang, Indeed, if running on 1 thread is fine, then using the standard v0.43.1 would work perfectly too, because the only reason for the forked kallisto is so v0.43.1 could run on multiple threads, so you would've gotten the exact same results anyway had the forked kallisto worked! This version was used because later versions would directly output to bam, making it not possible to analyze the result as it's being generated.

As long as in the output log, for each pseudoalignment after every "[quant] finding pseudoalignments for the reads ...", there's a "[quant] processed x reads, y reads pseudoaligned" (where x and y are numbers depending on the dataset), then it worked normally. The issue is when there isn't the latter message.

Let's leave this thread open as the mentioned issue with the forked kallisto is still there. We hypothesize it's due to different Linux systems, and hope to upload a fix soon.

Let me know if there's anything else with psirc I can help you with, including the meaning of the information in the output files. You can open another thread or send me a message, either one works.

Regards, Ken

Hi xjyx,

@xjyx

I meet the same problem as you did. Have you solved it?

Yours, Alipe

Hi Liu Peng,

Following the suggestion of the author, I replaced the kallisto static executable the author provided here with kallisto v0.43.1 and then I can ran the piepline normally.

Regards, Sui liang

Christina-hshi / psirc

Detected no BSJ using psirc_v1.0.pl #1