Issue with nanopore sequencing data on multiplex PCR

[marshelma]

Hi, I want to use SpecImmune to extract HLA types from nanopore data generated from multiplex PCR of HLA-I regions(The PCR protocol and primer sets for HLAs was found at https://onlinelibrary.wiley.com/doi/10.1111/tan.70164 )

Here is the command I use:

python3 scripts/main.py -r /home/xma/Dropbox/WD/Amplicon/30-1224458908/raw_fastq/293-ABC/293-ABC.fastq -i HLA -n 293T -o /home/xma/Dropbox/WD/Amplicon/30-1224458908/out -y nanopore

However, I got the error from running SpecImmune:

(SpecImmune) xma@xma-HP-Pavilion-x360-Convertible-15-cr0xxx:~/biosoft/SpecImmune$ python3 scripts/main.py -r /home/xma/Dropbox/WD/Amplicon/30-1224458908/raw_fastq/293-ABC/293-ABC.fastq -i HLA -n 293T -o /home/xma/Dropbox/WD/Amplicon/30-1224458908/out -y nanopore
{'r': '/home/xma/Dropbox/WD/Amplicon/30-1224458908/raw_fastq/293-ABC/293-ABC.fastq', 'n': '293T', 'o': '/home/xma/Dropbox/WD/Amplicon/30-1224458908/out', 'i': 'HLA', 'j': 5, 'k': 5, 'y': 'nanopore', 'db': '/home/xma/biosoft/SpecImmune/scripts/../db/', 'hg38': None, 'first_run': True, 'min_identity': 0.85, 'hete_p': 0.3, 'candidate_allele_num': 200, 'min_read_num': 2, 'max_read_num': 500, 'RNA_type': 'traditional', 'seq_tech': 'wgs', 'align_method_1': 'bwa', 'align_method_2': 'minimap2', 'iteration': 1, 'dv_sif': None, 'snv_tool': 'longshot', 'drug_recommendation': False, 'visualization': True, 'version': False}
read binning...
start read binning...
[E::bwa_idx_load_from_disk] fail to locate the index files
alignment done.
Traceback (most recent call last):
File "/home/xma/biosoft/SpecImmune/scripts/read_binning.py", line 234, in
pbin = Binning()
File "/home/xma/biosoft/SpecImmune/scripts/read_binning.py", line 113, in init
self.bamfile = pysam.AlignmentFile(self.sam, 'rb')
File "pysam/libcalignmentfile.pyx", line 747, in pysam.libcalignmentfile.AlignmentFile.cinit
File "pysam/libcalignmentfile.pyx", line 996, in pysam.libcalignmentfile.AlignmentFile._open
ValueError: file has no sequences defined (mode='rb') - is it SAM/BAM format? Consider opening with check_sq=False
start type HLA-DQA1 for 293T...

Traceback (most recent call last):
File "/home/xma/biosoft/SpecImmune/scripts/select_best_reference_alleleV2.py", line 656, in
main()
File "/home/xma/biosoft/SpecImmune/scripts/select_best_reference_alleleV2.py", line 534, in main
bam, depth_file, sort_depth_file = map2db(args, gene, my_db, my_folder, args["max_read_num"],args["align_method"])
File "/home/xma/biosoft/SpecImmune/scripts/alignment_modules.py", line 145, in map2db
sub_fastq = subsample_fastq(gene, read_num, my_folder)
File "/home/xma/biosoft/SpecImmune/scripts/alignment_modules.py", line 113, in subsample_fastq
read_count = cout_read_num(fastq)
File "/home/xma/biosoft/SpecImmune/scripts/alignment_modules.py", line 103, in cout_read_num
f = open(fastq, "rb")
FileNotFoundError: [Errno 2] No such file or directory: '/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Reads//HLA-DQA1.long_read.fq.gz'
iteration 1
Traceback (most recent call last):
File "/home/xma/biosoft/SpecImmune/scripts/get_2ref_align.py", line 256, in
main()
File "/home/xma/biosoft/SpecImmune/scripts/get_2ref_align.py", line 214, in main
read_hla_file(ref_file)
File "/home/xma/biosoft/SpecImmune/scripts/get_2ref_align.py", line 10, in read_hla_file
with open(filename, 'r') as file:
FileNotFoundError: [Errno 2] No such file or directory: '/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/293T.HLA.type.result.txt'
start variant pipeline ...
Processing homo HLA-DQA1
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DQA1.bam does not exist
Processing homo HLA-DRA
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DRA.bam does not exist
Processing homo HLA-N
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-N.bam does not exist
Processing homo HLA-DRB1
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DRB1.bam does not exist
Processing homo HLA-DQA2
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DQA2.bam does not exist
Processing homo HLA-V
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-V.bam does not exist
Processing homo HLA-R
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-R.bam does not exist
Processing homo HLA-K
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-K.bam does not exist
Processing homo HLA-S
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-S.bam does not exist
Processing homo HLA-DRB5
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DRB5.bam does not exist
Processing homo HLA-Y
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-Y.bam does not exist
Processing homo HLA-W
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-W.bam does not exist
Processing homo HLA-DOA
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DOA.bam does not exist
Processing homo HLA-J
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-J.bam does not exist
Processing homo HLA-DQB1
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DQB1.bam does not exist
Processing homo HFE
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HFE.bam does not exist
Processing homo HLA-U
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-U.bam does not exist
Processing homo HLA-DPB2
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DPB2.bam does not exist
Processing homo HLA-DOB
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DOB.bam does not exist
Processing homo HLA-DMA
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DMA.bam does not exist
Processing homo HLA-A
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-A.bam does not exist
Processing homo HLA-DRB4
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DRB4.bam does not exist
Processing homo HLA-DPB1
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DPB1.bam does not exist
Processing homo MICB
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//MICB.bam does not exist
Processing homo HLA-DRB3
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DRB3.bam does not exist
Processing homo MICA
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//MICA.bam does not exist
Processing homo TAP1
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//TAP1.bam does not exist
Processing homo HLA-F
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-F.bam does not exist
Processing homo HLA-DPA1
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DPA1.bam does not exist
Processing homo HLA-DMB
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DMB.bam does not exist
Processing homo HLA-L
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-L.bam does not exist
Processing homo HLA-C
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-C.bam does not exist
Processing homo HLA-P
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-P.bam does not exist
Processing homo HLA-DPA2
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DPA2.bam does not exist
Processing homo HLA-T
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-T.bam does not exist
Processing homo HLA-G
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-G.bam does not exist
Processing homo HLA-DQB2
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-DQB2.bam does not exist
Processing homo TAP2
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//TAP2.bam does not exist
Processing homo HLA-H
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-H.bam does not exist
Processing homo HLA-E
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-E.bam does not exist
Processing homo HLA-B
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Genes_step2//HLA-B.bam does not exist
Sequence is reconstructed, start annotation...
parameter: sample:293T dir:/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/Sequences/ pop:Unknown wxs:tgs G_nom:0 db:/home/xma/biosoft/SpecImmune/scripts/../db/
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-A/HLA-A
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-B/HLA-B
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-C/HLA-C
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DMA/HLA-DMA
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DMB/HLA-DMB
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DOA/HLA-DOA
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DOB/HLA-DOB
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DPA1/HLA-DPA1
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DPB1/HLA-DPB1
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DPB2/HLA-DPB2
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DQA1/HLA-DQA1
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DQB1/HLA-DQB1
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DRA/HLA-DRA
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DRB1/HLA-DRB1
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DRB3/HLA-DRB3
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DRB4/HLA-DRB4
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DRB5/HLA-DRB5
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-E/HLA-E
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-F/HLA-F
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-G/HLA-G
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-H/HLA-H
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-J/HLA-J
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-K/HLA-K
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-L/HLA-L
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-P/HLA-P
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-V/HLA-V
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DQA2/HLA-DQA2
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-DPA2/HLA-DPA2
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-N/HLA-N
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-S/HLA-S
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-T/HLA-T
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-U/HLA-U
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA-W/HLA-W
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/MICA/MICA
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/MICB/MICB
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/TAP1/TAP1
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/TAP2/TAP2
blast /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HFE/HFE
optimize typing results by balancing alignment length and identity
/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/293T.HLA.type.result.txt does not exist
Traceback (most recent call last):
File "/home/xma/biosoft/SpecImmune/scripts/refine_typing.py", line 392, in
output(record_best_match, record_one_allele, gene_list, result_file, my_db.version_info, record_all_match,read_num_dict)
File "/home/xma/biosoft/SpecImmune/scripts/refine_typing.py", line 221, in output
read_num, step1_alleles = read_num_dict[tag]
TypeError: 'int' object is not subscriptable
Finished.

        python3 /home/xma/biosoft/SpecImmune/scripts/remap.py 293T HLA /home/xma/Dropbox/WD/Amplicon/30-1224458908/out nanopore wgs traditional 5 /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA.full.fasta

Traceback (most recent call last):
File "/home/xma/biosoft/SpecImmune/scripts/remap.py", line 191, in
main()
File "/home/xma/biosoft/SpecImmune/scripts/remap.py", line 155, in main
read_hla_file(step1_result, step1_res_dict)
File "/home/xma/biosoft/SpecImmune/scripts/remap.py", line 25, in read_hla_file
with open(filename, 'r') as file:
FileNotFoundError: [Errno 2] No such file or directory: '/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/293T.HLA.type.result.txt'

        python3 /home/xma/biosoft/SpecImmune/scripts/remap_allele.py 293T HLA /home/xma/Dropbox/WD/Amplicon/30-1224458908/out nanopore wgs traditional 5 /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA.full.fasta

Traceback (most recent call last):
File "/home/xma/biosoft/SpecImmune/scripts/remap_allele.py", line 242, in
main()
File "/home/xma/biosoft/SpecImmune/scripts/remap_allele.py", line 198, in main
read_hla_file(step1_result, step1_res_dict)
File "/home/xma/biosoft/SpecImmune/scripts/remap_allele.py", line 32, in read_hla_file
with open(filename, 'r') as file:
FileNotFoundError: [Errno 2] No such file or directory: '/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/293T.HLA.type.result.txt'

        python3 /home/xma/biosoft/SpecImmune/scripts/visualization.py 293T HLA /home/xma/Dropbox/WD/Amplicon/30-1224458908/out nanopore wgs traditional 5 /home/xma/biosoft/SpecImmune/scripts/../db//HLA/HLA.full.fasta

Traceback (most recent call last):
File "/home/xma/biosoft/SpecImmune/scripts/visualization.py", line 382, in

main()

File "/home/xma/biosoft/SpecImmune/scripts/visualization.py", line 337, in main
read_hla_file(step1_result, step1_res_dict)
File "/home/xma/biosoft/SpecImmune/scripts/visualization.py", line 140, in read_hla_file
with open(filename, 'r') as file:
FileNotFoundError: [Errno 2] No such file or directory: '/home/xma/Dropbox/WD/Amplicon/30-1224458908/out/293T/293T.HLA.type.result.txt'

[wshuai294]

Hi, thanks for using SpecImmune! It looks like the database has not been constructed yet. Please make sure to run:

python scripts/make_db.py -o ./db -i HLA

before running the main script.
If you’ve already built the database, be sure to specify its path explicitly when running:

python main.py --db /path/to/db/

[marshelma]

Hi, thanks for using SpecImmune! It looks like the database has not been constructed yet. Please make sure to run:

python scripts/make_db.py -o ./db -i HLA

before running the main script. If you’ve already built the database, be sure to specify its path explicitly when running:

python main.py --db /path/to/db/

Thank you for the reply! It seems the bwa is missing, so the db is not constructed successfully.

[wshuai294]

It requires conda env. How it reports when you install the environment via the yml file?Best,Shuai WangOn Aug 19, 2025, at 7:42 PM, phenotype ***@***.***> wrote:marshelma left a comment (deepomicslab/SpecImmune#42) Hi, thanks for using SpecImmune! It looks like the database has not been constructed yet. Please make sure to run: python scripts/make_db.py -o ./db -i HLA before running the main script. If you’ve already built the database, be sure to specify its path explicitly when running: python main.py --db /path/to/db/ Thank you for the reply! It seems the bwa is missing, so the db is not constructed successfully. —Reply to this email directly, view it on GitHub, or unsubscribe.You are receiving this because you commented.Message ID: ***@***.***>

[marshelma]

It requires conda env. How it reports when you install the environment via the yml file?Best,Shuai WangOn Aug 19, 2025, at 7:42 PM, phenotype @.***> wrote:marshelma left a comment (deepomicslab/SpecImmune#42)

Hi, thanks for using SpecImmune! It looks like the database has not been constructed yet. Please make sure to run:
python scripts/make_db.py -o ./db -i HLA

before running the main script. If you’ve already built the database, be sure to specify its path explicitly when running:
python main.py --db /path/to/db/

Thank you for the reply! It seems the bwa is missing, so the db is not constructed successfully.

—Reply to this email directly, view it on GitHub, or unsubscribe.You are receiving this because you commented.Message ID: @.***>

The installation didn't report anything weird. When construct the db, it reported 'sh: 3: bwa: not found'. After install bwa, it works.

[troyeeee]

It requires conda env. How it reports when you install the environment via the yml file?Best,Shuai WangOn Aug 19, 2025, at 7:42 PM, phenotype @.> wrote:marshelma left a comment (deepomicslab/SpecImmune#42)
Hi, thanks for using SpecImmune! It looks like the database has not been constructed yet. Please make sure to run:
python scripts/make_db.py -o ./db -i HLA
before running the main script. If you’ve already built the database, be sure to specify its path explicitly when running:
python main.py --db /path/to/db/
Thank you for the reply! It seems the bwa is missing, so the db is not constructed successfully.
—Reply to this email directly, view it on GitHub, or unsubscribe.You are receiving this because you commented.Message ID: _@_.**>

The installation didn't report anything weird. When construct the db, it reported 'sh: 3: bwa: not found'. After install bwa, it works.

Thank you for reporting this. We've fixed the bug by adding bwa to the environment.yml file.