So many Error messages: please help

[RenaeAtkinson]

Directory ./ already exists. Writing into existing directory.. mkdir: cannot create directory ‘.//SCASA_testscasaHNVC02_20230414001259/’: File exists

Preparing for alignment.. Indexing reference.. Directory .//SCASA_testscasaHNVC02_20230414001259/0PRESETS//REF_INDEX/ already exists. Writing into existing directory.. Version Info: ### PLEASE UPGRADE SALMON ###

A newer version of salmon with important bug fixes and improvements is available.

The newest version, available at https://github.com/COMBINE-lab/salmon/releases contains new features, improvements, and bug fixes; please upgrade at your earliest convenience.

Sign up for the salmon mailing list to hear about new versions, features and updates at: https://oceangenomics.com/subscribe [2023-04-14 00:12:59.520] [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. [2023-04-14 00:12:59.520] [jLog] [info] building index out : .//SCASA_testscasaHNVC02_20230414001259/0PRESETS//REF_INDEX/ [2023-04-14 00:12:59.527] [puff::index::jointLog] [info] Running fixFasta

[Step 1 of 4] : counting k-mers

[2023-04-14 00:13:07.009] [puff::index::jointLog] [warning] Removed 236 transcripts that were sequence duplicates of indexed transcripts. [2023-04-14 00:13:07.010] [puff::index::jointLog] [warning] If you wish to retain duplicate transcripts, please use the --keepDuplicates flag [2023-04-14 00:13:07.012] [puff::index::jointLog] [info] Replaced 4 non-ATCG nucleotides [2023-04-14 00:13:07.012] [puff::index::jointLog] [info] Clipped poly-A tails from 11,186 transcripts wrote 76267 cleaned references [2023-04-14 00:13:07.789] [puff::index::jointLog] [info] Filter size not provided; estimating from number of distinct k-mers [2023-04-14 00:13:10.356] [puff::index::jointLog] [info] ntHll estimated 85097693 distinct k-mers, setting filter size to 2^31 Threads = 2 Vertex length = 31 Hash functions = 5 Filter size = 2147483648 Capacity = 2 Files: .//SCASA_testscasaHNVC02_20230414001259/0PRESETS//REF_INDEX/ref_k31_fixed.fa

Round 0, 0:2147483648 Pass Filling Filtering 1 36 77 2 5 0 True junctions count = 277411 False junctions count = 422333 Hash table size = 699744 Candidate marks count = 4646414

Reallocating bifurcations time: 0 True marks count: 3337299 Edges construction time: 6

Distinct junctions = 277411

TwoPaCo::buildGraphMain:: allocated with scalable_malloc; freeing. TwoPaCo::buildGraphMain:: Calling scalable_allocation_command(TBBMALLOC_CLEAN_ALL_BUFFERS, 0); allowedIn: 12 Max Junction ID: 318881 seen.size():2551057 kmerInfo.size():318882 approximateContigTotalLength: 66002535 counters for complex kmers: (prec>1 & succ>1)=26025 | (succ>1 & isStart)=63 | (prec>1 & isEnd)=73 | (isStart & isEnd)=10 contig count: 433949 element count: 98078572 complex nodes: 26171

of ones in rank vector: 433948

[2023-04-14 00:15:32.167] [puff::index::jointLog] [info] Starting the Pufferfish indexing by reading the GFA binary file. [2023-04-14 00:15:32.167] [puff::index::jointLog] [info] Setting the index/BinaryGfa directory .//SCASA_testscasaHNVC02_20230414001259/0PRESETS//REF_INDEX size = 98078572

| Loading contigs | Time = 47.228 ms

size = 98078572

| Loading contig boundaries | Time = 25.94 ms

Number of ones: 433948 Number of ones per inventory item: 512 Inventory entries filled: 848 433948 [2023-04-14 00:15:32.408] [puff::index::jointLog] [info] Done wrapping the rank vector with a rank9sel structure. [2023-04-14 00:15:32.412] [puff::index::jointLog] [info] contig count for validation: 433,948 [2023-04-14 00:15:32.736] [puff::index::jointLog] [info] Total # of Contigs : 433,948 [2023-04-14 00:15:32.736] [puff::index::jointLog] [info] Total # of numerical Contigs : 433,948 [2023-04-14 00:15:32.756] [puff::index::jointLog] [info] Total # of contig vec entries: 3,427,302 [2023-04-14 00:15:32.756] [puff::index::jointLog] [info] bits per offset entry 22 [2023-04-14 00:15:32.870] [puff::index::jointLog] [info] Done constructing the contig vector. 433949 [2023-04-14 00:15:33.302] [puff::index::jointLog] [info] # segments = 433,948 [2023-04-14 00:15:33.303] [puff::index::jointLog] [info] total length = 98,078,572 [2023-04-14 00:15:33.331] [puff::index::jointLog] [info] Reading the reference files ... [2023-04-14 00:15:34.093] [puff::index::jointLog] [info] positional integer width = 27 [2023-04-14 00:15:34.093] [puff::index::jointLog] [info] seqSize = 98,078,572 [2023-04-14 00:15:34.093] [puff::index::jointLog] [info] rankSize = 98,078,572 [2023-04-14 00:15:34.093] [puff::index::jointLog] [info] edgeVecSize = 0 [2023-04-14 00:15:34.093] [puff::index::jointLog] [info] num keys = 85,060,132 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 8 sec remaining: 0 min 0 sec Bitarray 445693632 bits (100.00 %) (array + ranks ) final hash 0 bits (0.00 %) (nb in final hash 0) [2023-04-14 00:15:41.958] [puff::index::jointLog] [info] mphf size = 53.1308 MB [2023-04-14 00:15:42.025] [puff::index::jointLog] [info] chunk size = 49,039,286 [2023-04-14 00:15:42.025] [puff::index::jointLog] [info] chunk 0 = [0, 49,039,286) [2023-04-14 00:15:42.025] [puff::index::jointLog] [info] chunk 1 = [49,039,286, 98,078,542) [2023-04-14 00:15:53.934] [puff::index::jointLog] [info] finished populating pos vector [2023-04-14 00:15:53.934] [puff::index::jointLog] [info] writing index components [2023-04-14 00:15:54.455] [puff::index::jointLog] [info] finished writing dense pufferfish index [2023-04-14 00:15:54.494] [jLog] [info] done building index Finnished indexing reference.. Begins pseudo-alignment.. nohup: redirecting stderr to stdout Congratulations! Pseudo-alignment has completed in 30 seconds! Scasa quantification has started.. Begin Scasa quantification for sample SRR10340946.. Error in file(con, "r") : cannot open the connection Calls: readLines -> file In addition: Warning message: In file(con, "r") : cannot open file './/SCASA_testscasaHNVC02_20230414001259/1ALIGN//SRR10340946_alignout/alevin/bfh.txt': No such file or directory Execution halted Loading required package: iterators Loading required package: parallel Error in readChar(con, 5L, useBytes = TRUE) : cannot open the connection Calls: load -> readChar In addition: Warning message: In readChar(con, 5L, useBytes = TRUE) : cannot open compressed file '/network/rit/lab/conklinlab/Renae/HNVC/HNVC02/SRR10340946/SCASA_testscasaHNVC02_20230414001259/2QUANT/SRR10340946_quant/Sample_eqClass.RData', probable reason 'No such file or directory' Execution halted Error in readChar(con, 5L, useBytes = TRUE) : cannot open the connection Calls: load -> readChar In addition: Warning message: In readChar(con, 5L, useBytes = TRUE) : cannot open compressed file './/SCASA_testscasaHNVC02_20230414001259/2QUANT//SRR10340946_quant//scasa_isoform_expression.RData', probable reason 'No such file or directory' Execution halted Congratulations! Scasa single cell RNA-Seq transcript quantification has completed in 30 seconds! All done!