error in assembling ONT DRS long reads

[nmrt-sahu]

I have used direct RNA-seq ONT long reads data. while running the rnabloom2, I got an error in the assembly step. I am not getting the reason behind it. could you help me out? I have pasted the command line output below for your reference.

rnabloom -long out.fastq -stranded -t 50 --uracil -outdir assambled
RNA-Bloom v2.0.1
args: [-long, out.fastq, -stranded, -t, THREADS, --uracil, -outdir, assambled]

ERROR: For input string: "THREADS"
java.lang.NumberFormatException: For input string: "THREADS"
at java.base/java.lang.NumberFormatException.forInputString(NumberFormatException.java:67)
at java.base/java.lang.Integer.parseInt(Integer.java:588)
at java.base/java.lang.Integer.parseInt(Integer.java:685)
at rnabloom.RNABloom.main(RNABloom.java:6433)
(rnabloom) [sklab202@compute-0-2 buffalo]$ rnabloom -long out.fastq -stranded -t 50 --uracil -outdir assambled
RNA-Bloom v2.0.1
args: [-long, out.fastq, -stranded, -t, 50, --uracil, -outdir, assambled]

name: rnabloom
outdir: assambled
WARNING: Output directory does not exist!
Created output directory at assambled

Turning on option -ntcard to count k-mers

K-mer counting with ntCard...
Running command: ntcard -t 50 -k 25 -c 65535 -p assambled/rnabloom @assambled/rnabloom.ntcard.readslist.txt...
Parsing histogram file assambled/rnabloom_k25.hist...
Unique k-mers (k=25): 2,415,168,253
Unique k-mers (k=25,c>1): 381,587,332
K-mer counting completed in 1m 11s

Bloom filters Memory (GB)

de Bruijn graph: 5.337153
k-mer counting: 6.745998

Total: 12.083151

Stage 1: Construct graph from reads (k=25)
Parsing out.fastq...
Parsed 5,572,535 sequences in 25m 13s
DBG Bloom filter FPR: 0.968 %
Counting Bloom filter FPR: 1.02 %
Stage 1 completed in 25m 30s

Stage 2: Correct long reads for "rnabloom"
Parsing out.fastq...
Corrected Read Lengths Sampling Distribution (n=10000)
min q1 med q3 max
56 449 624 1019 6622
Parsed 5,572,613 sequences.
Kept: 5,571,837 (100.0 %)
Discarded: 776 (0.0139 %)
Artifacts: 4 (7.1779614E-5%)
Corrected reads in 7m 8s
Extracting seed sequences...
strobemers: n=3, k=11, wMin=12, wMax=61, depth=3
Bloom filter FPR: 3.77 %
before: 5,532,596 after: 1,650,543 (29.8 %)
too short: 0
Extraction completed in 38m 6s
Stage 2 completed in 45m 15s

Stage 3: Assemble long reads for "rnabloom"
Overlapping sequences...
Parsed 0 overlap records in 0.001s
total reads: 1,650,543

• unique: 0 (0.0 %)
  • multi-seg: 0
    Unique reads extracted in 11.945s
    ERROR: Error extracting unique reads!
    ERROR: Error assembling long reads!

[kmnip]

For the first error, you forgot to specify an integer for number of threads. So, -t THREADS got errored out. I believed that you sorted out.

For the 2nd error, can you please check the log file name that looks something like this rnabloom.longreads.assembly.nr.fa.log? I think it had something to do with minimap2.