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Hi，
The error message "FASTQ files found in your input directory did not come in pairs! Please check and resubmit" occurs regardless of whether I use the test data you provided or other data
scasa --fastq Sample_01_S1_L001_R1_001.fastq, Sample_01_S1_L001_R2_001.fastq --ref /home/hfli/long_read_scRNA/Mrna/refMrna.fa --whitelist Sample_01_Whitelist.txt --nthreads 32
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SCASA V1.0.1
SINGLE CELL TRANSCRIPT QUANTIFICATION TOOL
Version Date: 2022-03-24
FOR ANY ISSUES, CONTACT: LU.PAN@KI.SE
https://github.com/eudoraleer/scasa/
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Directory ./ already exists. Writing into existing directory..
mkdir: 无法创建目录".//SCASA_My_Project_20240511164911/": 文件已存在
Preparing for alignment..
Indexing reference..
Directory .//SCASA_My_Project_20240511164911/0PRESETS//REF_INDEX/ already exists. Writing into existing directory..
Version Info: ### PLEASE UPGRADE SALMON ###
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contains new features, improvements, and bug fixes; please upgrade at your
earliest convenience.
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###[2024-05-11 16:49:12.158] [jLog] [warning] The salmon index is being built without any decoy sequences. It is recommended that decoy sequence (either computed auxiliary decoy sequence or the genome of the organism) be provided during indexing. Further details can be found at https://salmon.readthedocs.io/en/latest/salmon.html#preparing-transcriptome-indices-mapping-based-mode.
[2024-05-11 16:49:12.159] [jLog] [info] building index
out : .//SCASA_My_Project_20240511164911/0PRESETS//REF_INDEX/
[2024-05-11 16:49:12.159] [puff::index::jointLog] [info] Running fixFasta
[Step 1 of 4] : counting k-mers
[2024-05-11 16:49:20.266] [puff::index::jointLog] [warning] Removed 237 transcripts that were sequence duplicates of indexed transcripts.
[2024-05-11 16:49:20.266] [puff::index::jointLog] [warning] If you wish to retain duplicate transcripts, please use the --keepDuplicates flag
[2024-05-11 16:49:20.267] [puff::index::jointLog] [info] Replaced 5 non-ATCG nucleotides
[2024-05-11 16:49:20.267] [puff::index::jointLog] [info] Clipped poly-A tails from 12,501 transcripts
wrote 70629 cleaned references
[2024-05-11 16:49:21.041] [puff::index::jointLog] [info] Filter size not provided; estimating from number of distinct k-mers
[2024-05-11 16:49:23.797] [puff::index::jointLog] [info] ntHll estimated 84081876 distinct k-mers, setting filter size to 2^31
Threads = 2
Vertex length = 31
Hash functions = 5
Filter size = 2147483648
Capacity = 2
Files:
.//SCASA_My_Project_20240511164911/0PRESETS//REF_INDEX/ref_k31_fixed.fa
Round 0, 0:2147483648
Pass Filling Filtering
1 33 79
2 5 0
True junctions count = 266516
False junctions count = 404655
Hash table size = 671171
Candidate marks count = 4091391
Reallocating bifurcations time: 0
True marks count: 2954071
Edges construction time: 5
Distinct junctions = 266516
allowedIn: 12
Max Junction ID: 308126
seen.size():2465017 kmerInfo.size():308127
approximateContigTotalLength: 65012593
counters for complex kmers:
(prec>1 & succ>1)=25336 | (succ>1 & isStart)=72 | (prec>1 & isEnd)=60 | (isStart & isEnd)=10
contig count: 417773 element count: 96576272 complex nodes: 25478
of ones in rank vector: 417772
[2024-05-11 16:51:40.180] [puff::index::jointLog] [info] Starting the Pufferfish indexing by reading the GFA binary file.
[2024-05-11 16:51:40.180] [puff::index::jointLog] [info] Setting the index/BinaryGfa directory .//SCASA_My_Project_20240511164911/0PRESETS//REF_INDEX
size = 96576272
| Loading contigs | Time = 21.717 ms
size = 96576272
| Loading contig boundaries | Time = 10.621 ms
Number of ones: 417772
Number of ones per inventory item: 512
Inventory entries filled: 816
417772
[2024-05-11 16:51:40.405] [puff::index::jointLog] [info] Done wrapping the rank vector with a rank9sel structure.
[2024-05-11 16:51:40.415] [puff::index::jointLog] [info] contig count for validation: 417,772
[2024-05-11 16:51:40.603] [puff::index::jointLog] [info] Total # of Contigs : 417,772
[2024-05-11 16:51:40.603] [puff::index::jointLog] [info] Total # of numerical Contigs : 417,772
[2024-05-11 16:51:40.662] [puff::index::jointLog] [info] Total # of contig vec entries: 3,035,777
[2024-05-11 16:51:40.662] [puff::index::jointLog] [info] bits per offset entry 22
[2024-05-11 16:51:40.816] [puff::index::jointLog] [info] Done constructing the contig vector. 417773
[2024-05-11 16:51:40.925] [puff::index::jointLog] [info] # segments = 417,772
[2024-05-11 16:51:40.925] [puff::index::jointLog] [info] total length = 96,576,272
[2024-05-11 16:51:40.993] [puff::index::jointLog] [info] Reading the reference files ...
[2024-05-11 16:51:41.701] [puff::index::jointLog] [info] positional integer width = 27
[2024-05-11 16:51:41.702] [puff::index::jointLog] [info] seqSize = 96,576,272
[2024-05-11 16:51:41.702] [puff::index::jointLog] [info] rankSize = 96,576,272
[2024-05-11 16:51:41.702] [puff::index::jointLog] [info] edgeVecSize = 0
[2024-05-11 16:51:41.702] [puff::index::jointLog] [info] num keys = 84,043,112
for info, total work write each : 2.331 total work inram from level 3 : 4.322 total work raw : 25.000
[Building BooPHF] 100 % elapsed: 0 min 14 sec remaining: 0 min 0 sec
Bitarray 440364608 bits (100.00 %) (array + ranks )
final hash 0 bits (0.00 %) (nb in final hash 0)
[2024-05-11 16:51:56.145] [puff::index::jointLog] [info] mphf size = 52.4956 MB
[2024-05-11 16:51:56.356] [puff::index::jointLog] [info] chunk size = 48,288,136
[2024-05-11 16:51:56.356] [puff::index::jointLog] [info] chunk 0 = [0, 48,288,136)
[2024-05-11 16:51:56.356] [puff::index::jointLog] [info] chunk 1 = [48,288,136, 96,576,242)
[2024-05-11 16:52:13.341] [puff::index::jointLog] [info] finished populating pos vector
[2024-05-11 16:52:13.342] [puff::index::jointLog] [info] writing index components
[2024-05-11 16:52:13.720] [puff::index::jointLog] [info] finished writing dense pufferfish index
[2024-05-11 16:52:13.854] [jLog] [info] done building index
Finnished indexing reference..
Begins pseudo-alignment..
FASTQ files found in your input directory did not come in pairs! Please check and resubmit.