# No species were selected from the prescreen. Because of this the custom ChocoPhlAn database is empty. This will result in zero species-specific gene families and pathways **URL:** \ **Category:** HUMAnN\ **Created:** [July 23, 2020, 5:06pm UTC](https://forum.biobakery.org/t/no-species-were-selected-from-the-prescreen-because-of-this-the-custom-chocophlan-database-is-empty-this-will-result-in-zero-species-specific-gene-families-and-pathways/765 "2020-07-23T17:06:05Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![Jamie\_Chou](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/jamie_chou/32/325_2.png) [@Jamie\_Chou](https://forum.biobakery.org/u/Jamie_Chou)\ **Post date:** [July 23, 2020, 5:06pm UTC](https://forum.biobakery.org/t/no-species-were-selected-from-the-prescreen-because-of-this-the-custom-chocophlan-database-is-empty-this-will-result-in-zero-species-specific-gene-families-and-pathways/765/1 "2020-07-23T17:06:05Z")
07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Running humann2 v2.8.1 07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Output files will be written to: /home/bio/temp 07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Writing temp files to directory: /home/bio/temp/AH1\_humann2\_temp 07/24/2020 12:54:38 AM - humann2.utilities - INFO: File ( /home/bio/temp/concat/AH1.fq ) is of format: fastq 07/24/2020 12:54:38 AM - humann2.utilities - DEBUG: Check software, bowtie2, for required version, 2.2 07/24/2020 12:54:38 AM - humann2.utilities - INFO: Using bowtie2 version 2.3 07/24/2020 12:54:38 AM - humann2.humann2 - INFO: Search mode set to uniref90 because a uniref90 translated search database is selected 07/24/2020 12:54:38 AM - humann2.utilities - DEBUG: Check software, diamond, for required version, 0.8.22 07/24/2020 12:54:38 AM - humann2.utilities - INFO: Using diamond version 0.8.36 07/24/2020 12:54:38 AM - humann2.config - INFO: Run config settings: DATABASE SETTINGS nucleotide database folder = /home/bio/db/humann2/chocophlan protein database folder = /home/bio/db/humann2 pathways database file 1 = /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc\_reactions\_level4ec\_only.uniref.bz2 pathways database file 2 = /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc\_pathways\_structured\_filtered utility mapping database folder = /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/misc RUN MODES resume = False verbose = False bypass prescreen = False bypass nucleotide index = False bypass nucleotide search = False bypass translated search = False translated search = diamond pick frames = off threads = 88 SEARCH MODE search mode = uniref90 identity threshold = 90.0 ALIGNMENT SETTINGS evalue threshold = 1.0 prescreen threshold = 0.01 translated subject coverage threshold = 50.0 translated query coverage threshold = 90.0 PATHWAYS SETTINGS minpath = on xipe = off gap fill = on INPUT AND OUTPUT FORMATS input file format = fastq output file format = tsv output max decimals = 10 remove stratified output = False remove column description output = False log level = DEBUG 07/24/2020 12:54:38 AM - humann2.store - DEBUG: Initialize Alignments class instance to minimize memory use 07/24/2020 12:54:38 AM - humann2.store - DEBUG: Initialize Reads class instance to minimize memory use 07/24/2020 12:54:44 AM - humann2.humann2 - INFO: Load pathways database part 1: /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc\_reactions\_level4ec\_only.uniref.bz2 07/24/2020 12:54:56 AM - humann2.humann2 - INFO: Load pathways database part 2: /home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/humann2/data/pathways/metacyc\_pathways\_structured\_filtered 07/24/2020 12:54:56 AM - humann2.utilities - DEBUG: Check software, metaphlan2.py, for required version, 2.6 07/24/2020 12:54:57 AM - humann2.utilities - INFO: Using metaphlan2.py version 2.7 07/24/2020 12:54:57 AM - humann2.search.prescreen - INFO: Running metaphlan2.py … 07/24/2020 12:54:57 AM - humann2.utilities - DEBUG: Using software: /home/bio/miniconda3/envs/meta/bin/metaphlan2.py 07/24/2020 12:54:57 AM - humann2.utilities - INFO: Execute command: /home/bio/miniconda3/envs/meta/bin/metaphlan2.py /home/bio/temp/concat/AH1.fq -t rel\_ab -o /home/bio/temp/AH1\_humann2\_temp/AH1\_metaphlan\_bugs\_list.tsv --input\_type multifastq --bowtie2out /home/bio/temp/AH1\_humann2\_temp/AH1\_metaphlan\_bowtie2.txt --nproc 88 07/24/2020 12:56:03 AM - humann2.utilities - DEBUG: Help message for read\_fastx.py Traceback (most recent call last): File “/home/bio/miniconda3/envs/meta/bin/read\_fastx.py”, line 123, in read\_and\_write\_raw(f, opened=False, min\_len=min\_len) File “/home/bio/miniconda3/envs/meta/bin/read\_fastx.py”, line 89, in read\_and\_write\_raw read\_and\_write\_raw\_int(inf, min\_len=min\_len) File “/home/bio/miniconda3/envs/meta/bin/read\_fastx.py”, line 74, in read\_and\_write\_raw\_int for record in SeqIO.parse(fd, fmt): File “/home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/Bio/SeqIO/ **init**.py”, line 600, in parse for r in i: File “/home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/Bio/SeqIO/QualityIO.py”, line 1031, in FastqPhredIterator for title\_line, seq\_string, quality\_string in FastqGeneralIterator(handle): File “/home/bio/miniconda3/envs/meta/lib/python2.7/site-packages/Bio/SeqIO/QualityIO.py”, line 951, in FastqGeneralIterator % (title\_line, seq\_len, len(quality\_string))) ValueError: Lengths of sequence and quality values differs for A00920:121:HGNNYDRXX:2:2278:12391:36918:N:0:TACAAGTA+AGTCAACA#0/1 (250 and 1229240). 07/24/2020 12:56:03 AM - humann2.humann2 - INFO: TIMESTAMP: Completed prescreen : 67 seconds 07/24/2020 12:56:03 AM - humann2.search.prescreen - INFO: Total species selected from prescreen: 0 07/24/2020 12:56:03 AM - humann2.search.prescreen - DEBUG: No species were selected from the prescreen. Because of this the custom ChocoPhlAn database is empty. This will result in zero species-specific gene families and pathways. 07/24/2020 12:56:03 AM - humann2.humann2 - INFO: TIMESTAMP: Completed custom database creation : 0 seconds 07/24/2020 12:56:03 AM - humann2.humann2 - DEBUG: Custom database is empty 07/24/2020 12:56:03 AM - humann2.store - DEBUG: Initialize Reads class instance to minimize memory use 07/24/2020 12:56:09 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v/tmpZDlKOt 07/24/2020 12:56:09 AM - humann2.search.translated - DEBUG: Convert unaligned reads fastq file to fasta 07/24/2020 12:56:14 AM - humann2.search.translated - INFO: Running diamond … 07/24/2020 12:56:14 AM - humann2.search.translated - INFO: Aligning to reference database: uniref90\_annotated.1.1.dmnd 07/24/2020 12:56:14 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v/diamond\_m8\_DyEiWu 07/24/2020 12:56:14 AM - humann2.utilities - DEBUG: Using software: /home/bio/miniconda3/envs/meta/bin/diamond 07/24/2020 12:56:14 AM - humann2.utilities - INFO: Execute command: /home/bio/miniconda3/envs/meta/bin/diamond blastx --query /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v/tmp2Emh\_m --evalue 1.0 --threads 88 --max-target-seqs 20 --outfmt 6 --db /home/bio/db/humann2/uniref90\_annotated.1.1 --out /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v/diamond\_m8\_DyEiWu --tmpdir /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v 07/24/2020 01:01:10 AM - humann2.utilities - DEBUG: diamond v0.8.36.98 | by Benjamin Buchfink [buchfink@gmail.com](mailto:buchfink@gmail.com) Check [http://github.com/bbuchfink/diamond](http://github.com/bbuchfink/diamond) for updates. #CPU threads: 88 Scoring parameters: (Matrix=BLOSUM62 Lambda=0.267 K=0.041 Penalties=11/1) #Target sequences to report alignments for: 20 Temporary directory: /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v Opening the database… [9e-06s] Opening the input file… [1.4e-05s] Opening the output file… [1.1e-05s] Loading query sequences… [0.94992s] Running complexity filter… [0.643231s] Building query histograms… [0.181994s] Allocating buffers… [0.004273s] Loading reference sequences… [3.11789s] Building reference histograms… [2.1769s] Allocating buffers… [0.004019s] Initializing temporary storage… [0.01668s] Processing query chunk 0, reference chunk 0, shape 0, index chunk 0. Building reference index… [2.59516s] Building query index… [0.148841s] Building seed filter… [0.258946s] Searching alignments… [0.892207s] Processing query chunk 0, reference chunk 0, shape 0, index chunk 1. Building reference index… [2.47919s] Building query index… [0.106385s] Building seed filter… [0.252801s] Searching alignments… [0.70194s] Processing query chunk 0, reference chunk 0, shape 0, index chunk 2. Building reference index… [2.56884s] Building query index… [0.119807s] Building seed filter… [0.335353s] Searching alignments… [0.746279s] Processing query chunk 0, reference chunk 0, shape 0, index chunk 3. Building reference index… [2.45842s] Building query index… [0.08127s] Building seed filter… [0.346824s] Searching alignments… [1.218s] Processing query chunk 0, reference chunk 0, shape 1, index chunk 0. 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Building reference index… [2.6507s] Building query index… [0.095286s] Building seed filter… [0.315834s] Searching alignments… [0.740181s] Processing query chunk 0, reference chunk 0, shape 2, index chunk 1. Building reference index… [2.82734s] Building query index… [0.09418s] Building seed filter… [0.339952s] Searching alignments… [1.12977s] Processing query chunk 0, reference chunk 0, shape 2, index chunk 2. Building reference index… [2.95134s] Building query index… [0.09651s] Building seed filter… [0.366048s] Searching alignments… [0.734481s] Processing query chunk 0, reference chunk 0, shape 2, index chunk 3. Building reference index… [2.88367s] Building query index… [0.100285s] Building seed filter… [0.438955s] Searching alignments… [0.84678s] Processing query chunk 0, reference chunk 0, shape 3, index chunk 0. Building reference index… [2.98181s] Building query index… [0.086555s] Building seed filter… [0.408336s] Searching alignments… [0.595884s] Processing query chunk 0, reference chunk 0, shape 3, index chunk 1. Building reference index… [3.1383s] Building query index… [0.102204s] Building seed filter… [0.361227s] Searching alignments… [0.694366s] Processing query chunk 0, reference chunk 0, shape 3, index chunk 2. Building reference index… [3.14952s] Building query index… [0.095599s] Building seed filter… [0.390542s] Searching alignments… [0.77369s] Processing query chunk 0, reference chunk 0, shape 3, index chunk 3. Building reference index… [3.08838s] Building query index… [0.095059s] Building seed filter… [0.39518s] Searching alignments… [0.919213s] Deallocating buffers… [0.294269s] Opening temporary output file… [0.000104s] Computing alignments… [2.42973s] Deallocating reference… [0.103076s] Loading reference sequences… [4.38223s] Building reference histograms… [2.41481s] Allocating buffers… [0.004017s] Initializing temporary storage… [0.015365s] Processing query chunk 0, reference chunk 1, shape 0, index chunk 0. Building reference index… [2.3863s] Building query index… [0.128065s] Building seed filter… [0.263486s] Searching alignments… [0.870244s] Processing query chunk 0, reference chunk 1, shape 0, index chunk 1. Building reference index… [2.44523s] Building query index… [0.087284s] Building seed filter… [0.247231s] Searching alignments… [0.97806s] Processing query chunk 0, reference chunk 1, shape 0, index chunk 2. Building reference index… [2.55059s] Building query index… [0.112499s] Building seed filter… [0.256802s] Searching alignments… [0.943185s] Processing query chunk 0, reference chunk 1, shape 0, index chunk 3. Building reference index… [2.33231s] Building query index… [0.106547s] Building seed filter… [0.263331s] Searching alignments… [0.881897s] Processing query chunk 0, reference chunk 1, shape 1, index chunk 0. Building reference index… [2.39147s] Building query index… [0.10137s] Building seed filter… [0.332774s] Searching alignments… [0.826884s] Processing query chunk 0, reference chunk 1, shape 1, index chunk 1. Building reference index… [2.57037s] Building query index… [0.082806s] Building seed filter… [0.34644s] Searching alignments… [0.743145s] Processing query chunk 0, reference chunk 1, shape 1, index chunk 2. Building reference index… [2.59866s] Building query index… [0.087607s] Building seed filter… [0.29012s] Searching alignments… [1.04797s] Processing query chunk 0, reference chunk 1, shape 1, index chunk 3. Building reference index… [2.49335s] Building query index… [0.095637s] Building seed filter… [0.284832s] Searching alignments… [0.786772s] Processing query chunk 0, reference chunk 1, shape 2, index chunk 0. Building reference index… [2.55282s] Building query index… [0.088018s] Building seed filter… [0.277688s] Searching alignments… [0.831547s] Processing query chunk 0, reference chunk 1, shape 2, index chunk 1. Building reference index… [2.6731s] Building query index… [0.073242s] Building seed filter… [0.386872s] Searching alignments… [0.683937s] Processing query chunk 0, reference chunk 1, shape 2, index chunk 2. Building reference index… [2.83754s] Building query index… [0.085035s] Building seed filter… [0.449488s] Searching alignments… [1.24259s] Processing query chunk 0, reference chunk 1, shape 2, index chunk 3. Building reference index… [2.96462s] Building query index… [0.082029s] Building seed filter… [0.446799s] Searching alignments… [0.569426s] Processing query chunk 0, reference chunk 1, shape 3, index chunk 0. Building reference index… [2.99553s] Building query index… [0.081565s] Building seed filter… [0.399137s] Searching alignments… [0.678737s] Processing query chunk 0, reference chunk 1, shape 3, index chunk 1. Building reference index… [3.20053s] Building query index… [0.077606s] Building seed filter… [0.369711s] Searching alignments… [0.573682s] Processing query chunk 0, reference chunk 1, shape 3, index chunk 2. Building reference index… [3.15797s] Building query index… [0.100836s] Building seed filter… [0.344765s] Searching alignments… [0.767472s] Processing query chunk 0, reference chunk 1, shape 3, index chunk 3. Building reference index… [2.99569s] Building query index… [0.098234s] Building seed filter… [0.376472s] Searching alignments… [0.638577s] Deallocating buffers… [0.275702s] Opening temporary output file… [0.000115s] Computing alignments… [1.88496s] Deallocating reference… [0.134175s] Loading reference sequences… [3.10932s] Building reference histograms… [1.97341s] Allocating buffers… [0.004945s] Initializing temporary storage… [0.015538s] Processing query chunk 0, reference chunk 2, shape 0, index chunk 0. Building reference index… [2.49894s] Building query index… [0.10634s] Building seed filter… [0.287296s] Searching alignments… [0.729471s] Processing query chunk 0, reference chunk 2, shape 0, index chunk 1. Building reference index… [2.54813s] Building query index… [0.10915s] Building seed filter… [0.282037s] Searching alignments… [0.838157s] Processing query chunk 0, reference chunk 2, shape 0, index chunk 2. Building reference index… [2.58657s] Building query index… [0.11746s] Building seed filter… [0.269336s] Searching alignments… [1.0608s] Processing query chunk 0, reference chunk 2, shape 0, index chunk 3. Building reference index… [2.48552s] Building query index… [0.107657s] Building seed filter… [0.28429s] Searching alignments… [0.893738s] Processing query chunk 0, reference chunk 2, shape 1, index chunk 0. Building reference index… [2.49033s] Building query index… [0.097985s] Building seed filter… [0.22968s] Searching alignments… [0.827066s] Processing query chunk 0, reference chunk 2, shape 1, index chunk 1. Building reference index… [2.58708s] Building query index… [0.104543s] Building seed filter… [0.293924s] Searching alignments… [0.660024s] Processing query chunk 0, reference chunk 2, shape 1, index chunk 2. Building reference index… [2.60007s] Building query index… [0.11483s] Building seed filter… [0.252577s] Searching alignments… [0.719922s] Processing query chunk 0, reference chunk 2, shape 1, index chunk 3. Building reference index… [2.43576s] Building query index… [0.094867s] Building seed filter… [0.287645s] Searching alignments… [0.701374s] Processing query chunk 0, reference chunk 2, shape 2, index chunk 0. Building reference index… [2.34868s] Building query index… [0.089875s] Building seed filter… [0.25751s] Searching alignments… [0.799089s] Processing query chunk 0, reference chunk 2, shape 2, index chunk 1. Building reference index… [2.49723s] Building query index… [0.096167s] Building seed filter… [0.277589s] Searching alignments… [0.823164s] Processing query chunk 0, reference chunk 2, shape 2, index chunk 2. Building reference index… [2.61548s] Building query index… [0.101088s] Building seed filter… [0.265914s] Searching alignments… [0.791204s] Processing query chunk 0, reference chunk 2, shape 2, index chunk 3. Building reference index… [2.42162s] Building query index… [0.093691s] Building seed filter… [0.253444s] Searching alignments… [0.679295s] Processing query chunk 0, reference chunk 2, shape 3, index chunk 0. Building reference index… [2.31967s] Building query index… [0.100062s] Building seed filter… [0.3329s] Searching alignments… [0.628405s] Processing query chunk 0, reference chunk 2, shape 3, index chunk 1. Building reference index… [2.56571s] Building query index… [0.084137s] Building seed filter… [0.222692s] Searching alignments… [0.718503s] Processing query chunk 0, reference chunk 2, shape 3, index chunk 2. Building reference index… [2.56744s] Building query index… [0.09302s] Building seed filter… [0.273627s] Searching alignments… [0.507588s] Processing query chunk 0, reference chunk 2, shape 3, index chunk 3. Building reference index… [2.39665s] Building query index… [0.096917s] Building seed filter… [0.271391s] Searching alignments… [0.664346s] Deallocating buffers… [0.319991s] Opening temporary output file… [0.00011s] Computing alignments… [2.33864s] Deallocating reference… [0.133355s] Loading reference sequences… [3.40252s] Building reference histograms… [2.07839s] Allocating buffers… [0.004179s] Initializing temporary storage… [0.015844s] Processing query chunk 0, reference chunk 3, shape 0, index chunk 0. Building reference index… [2.78663s] Building query index… [0.101486s] Building seed filter… [0.324625s] Searching alignments… [1.25771s] Processing query chunk 0, reference chunk 3, shape 0, index chunk 1. Building reference index… [2.84048s] Building query index… [0.108914s] Building seed filter… [0.33605s] Searching alignments… [1.01265s] Processing query chunk 0, reference chunk 3, shape 0, index chunk 2. Building reference index… [2.80122s] Building query index… [0.089795s] Building seed filter… [0.285051s] Searching alignments… [0.940518s] Processing query chunk 0, reference chunk 3, shape 0, index chunk 3. Building reference index… [2.71729s] Building query index… [0.109229s] Building seed filter… [0.299107s] Searching alignments… [0.748951s] Processing query chunk 0, reference chunk 3, shape 1, index chunk 0. Building reference index… [2.61764s] Building query index… [0.096895s] Building seed filter… [0.265124s] Searching alignments… [0.826025s] Processing query chunk 0, reference chunk 3, shape 1, index chunk 1. Building reference index… [2.78608s] Building query index… [0.108095s] Building seed filter… [0.289734s] Searching alignments… [0.863198s] Processing query chunk 0, reference chunk 3, shape 1, index chunk 2. Building reference index… [2.82005s] Building query index… [0.101616s] Building seed filter… [0.311856s] Searching alignments… [0.794061s] Processing query chunk 0, reference chunk 3, shape 1, index chunk 3. Building reference index… [2.61539s] Building query index… [0.096688s] Building seed filter… [0.331517s] Searching alignments… [0.733005s] Processing query chunk 0, reference chunk 3, shape 2, index chunk 0. Building reference index… [2.60875s] Building query index… [0.093445s] Building seed filter… [0.312869s] Searching alignments… [0.675556s] Processing query chunk 0, reference chunk 3, shape 2, index chunk 1. Building reference index… [2.73663s] Building query index… [0.108951s] Building seed filter… [0.307627s] Searching alignments… [0.647167s] Processing query chunk 0, reference chunk 3, shape 2, index chunk 2. Building reference index… [2.67541s] Building query index… [0.108586s] Building seed filter… [0.281827s] Searching alignments… [0.685273s] Processing query chunk 0, reference chunk 3, shape 2, index chunk 3. Building reference index… [2.63533s] Building query index… [0.102384s] Building seed filter… [0.282545s] Searching alignments… [0.833469s] Processing query chunk 0, reference chunk 3, shape 3, index chunk 0. Building reference index… [2.54113s] Building query index… [0.096377s] Building seed filter… [0.298138s] Searching alignments… [0.75816s] Processing query chunk 0, reference chunk 3, shape 3, index chunk 1. Building reference index… [2.67781s] Building query index… [0.09915s] Building seed filter… [0.297354s] Searching alignments… [0.72485s] Processing query chunk 0, reference chunk 3, shape 3, index chunk 2. Building reference index… [2.67581s] Building query index… [0.104657s] Building seed filter… [0.276573s] Searching alignments… [0.59556s] Processing query chunk 0, reference chunk 3, shape 3, index chunk 3. Building reference index… [2.57122s] Building query index… [0.095354s] Building seed filter… [0.280215s] Searching alignments… [0.720123s] Deallocating buffers… [0.329535s] Opening temporary output file… [0.000132s] Computing alignments… [2.51166s] Deallocating reference… [0.148843s] Loading reference sequences… [0.163781s] Building reference histograms… [0.188441s] Allocating buffers… [0.004698s] Initializing temporary storage… [0.019274s] Processing query chunk 0, reference chunk 4, shape 0, index chunk 0. Building reference index… [0.160548s] Building query index… [0.079011s] Building seed filter… [0.034525s] Searching alignments… [0.121273s] Processing query chunk 0, reference chunk 4, shape 0, index chunk 1. Building reference index… [0.159379s] Building query index… [0.113094s] Building seed filter… [0.033118s] Searching alignments… [0.131446s] Processing query chunk 0, reference chunk 4, shape 0, index chunk 2. Building reference index… [0.149408s] Building query index… [0.101582s] Building seed filter… [0.034058s] Searching alignments… [0.52861s] Processing query chunk 0, reference chunk 4, shape 0, index chunk 3. Building reference index… [0.129733s] Building query index… [0.105485s] Building seed filter… [0.036182s] Searching alignments… [0.518204s] Processing query chunk 0, reference chunk 4, shape 1, index chunk 0. Building reference index… [0.127096s] Building query index… [0.09089s] Building seed filter… [0.035789s] Searching alignments… [0.045675s] Processing query chunk 0, reference chunk 4, shape 1, index chunk 1. Building reference index… [0.135742s] Building query index… [0.101391s] Building seed filter… [0.033177s] Searching alignments… [0.155112s] Processing query chunk 0, reference chunk 4, shape 1, index chunk 2. Building reference index… [0.147998s] Building query index… [0.100192s] Building seed filter… [0.036285s] Searching alignments… [0.442196s] Processing query chunk 0, reference chunk 4, shape 1, index chunk 3. Building reference index… [0.134996s] Building query index… [0.095986s] Building seed filter… [0.032232s] Searching alignments… [0.2171s] Processing query chunk 0, reference chunk 4, shape 2, index chunk 0. Building reference index… [0.139522s] Building query index… [0.095769s] Building seed filter… [0.031706s] Searching alignments… [0.346017s] Processing query chunk 0, reference chunk 4, shape 2, index chunk 1. Building reference index… [0.151409s] Building query index… [0.099187s] Building seed filter… [0.031547s] Searching alignments… [0.281806s] Processing query chunk 0, reference chunk 4, shape 2, index chunk 2. Building reference index… [0.143743s] Building query index… [0.096926s] Building seed filter… [0.03628s] Searching alignments… [0.064184s] Processing query chunk 0, reference chunk 4, shape 2, index chunk 3. Building reference index… [0.131663s] Building query index… [0.089608s] Building seed filter… [0.033461s] Searching alignments… [0.033455s] Processing query chunk 0, reference chunk 4, shape 3, index chunk 0. Building reference index… [0.129791s] Building query index… [0.0971s] Building seed filter… [0.032715s] Searching alignments… [0.081845s] Processing query chunk 0, reference chunk 4, shape 3, index chunk 1. Building reference index… [0.136792s] Building query index… [0.095205s] Building seed filter… [0.032075s] Searching alignments… [0.037459s] Processing query chunk 0, reference chunk 4, shape 3, index chunk 2. Building reference index… [0.144873s] Building query index… [0.097941s] Building seed filter… [0.032173s] Searching alignments… [0.402392s] Processing query chunk 0, reference chunk 4, shape 3, index chunk 3. Building reference index… [0.131428s] Building query index… [0.088914s] Building seed filter… [0.031901s] Searching alignments… [0.040683s] Deallocating buffers… [0.016488s] Opening temporary output file… [0.000112s] Computing alignments… [0.458893s] Deallocating reference… [0.007812s] Loading reference sequences… [2.3e-05s] Deallocating buffers… [0.013108s] Joining output blocks… [4.07241s] Deallocating queries… [0.007177s] Loading query sequences… [2.5e-05s] Closing the input file… [4e-06s] Closing the output file… [1.3e-05s] Closing the database file… [3e-06s] Total time = 295.506s Reported 5541065 pairwise alignments, 5541065 HSSPs. 299682 queries aligned. 07/24/2020 01:01:10 AM - humann2.utilities - DEBUG: Using software: /usr/bin/cat 07/24/2020 01:01:10 AM - humann2.utilities - INFO: Execute command: /usr/bin/cat /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v/diamond\_m8\_DyEiWu 07/24/2020 01:01:10 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v/tmp2Emh\_m 07/24/2020 01:01:10 AM - humann2.humann2 - INFO: TIMESTAMP: Completed translated alignment : 308 seconds 07/24/2020 01:02:10 AM - humann2.search.blastx\_coverage - INFO: Total alignments without coverage information: 0 07/24/2020 01:02:10 AM - humann2.search.blastx\_coverage - INFO: Total proteins in blastx output: 101 07/24/2020 01:02:10 AM - humann2.search.blastx\_coverage - INFO: Total proteins without lengths: 0 07/24/2020 01:02:10 AM - humann2.search.blastx\_coverage - INFO: Proteins with coverage greater than threshold (50.0): 58 07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total alignments where percent identity is not a number: 0 07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total alignments where alignment length is not a number: 0 07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total alignments where E-value is not a number: 0 07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total translated alignments not included based on large e-value: 0 07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total translated alignments not included based on small percent identity: 5149438 07/24/2020 01:03:13 AM - humann2.utilities - DEBUG: Total translated alignments not included based on small query coverage: 4094022 07/24/2020 01:03:13 AM - humann2.search.translated - DEBUG: Total translated alignments not included based on small subject coverage value: 4634 07/24/2020 01:03:19 AM - humann2.utilities - DEBUG: Remove file: /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v/tmpgPxyDE 07/24/2020 01:03:19 AM - humann2.humann2 - INFO: TIMESTAMP: Completed translated alignment post-processing : 129 seconds 07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Total bugs after translated alignment: 1 07/24/2020 01:03:19 AM - humann2.humann2 - INFO: unclassified: 42862 hits 07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Total gene families after translated alignment: 58 07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Unaligned reads after translated alignment: 92.4778511558 % 07/24/2020 01:03:19 AM - humann2.humann2 - INFO: Computing gene families … 07/24/2020 01:03:19 AM - humann2.quantify.families - DEBUG: Compute gene families 07/24/2020 01:03:19 AM - humann2.store - INFO: Total gene families : 58 unclassified : 58 gene families 07/24/2020 01:03:40 AM - humann2.humann2 - INFO: TIMESTAMP: Completed computing gene families : 21 seconds 07/24/2020 01:03:40 AM - humann2.humann2 - INFO: Computing pathways abundance and coverage … 07/24/2020 01:03:40 AM - humann2.quantify.modules - DEBUG: Write flat reactions to pathways file for Minpath 07/24/2020 01:03:40 AM - humann2.quantify.modules - INFO: Compute reaction scores for bug: unclassified 07/24/2020 01:03:41 AM - humann2.quantify.modules - INFO: Compute reaction scores for bug: all 07/24/2020 01:03:41 AM - humann2.quantify.modules - DEBUG: Print pathways \_Abundance 07/24/2020 01:03:41 AM - humann2.quantify.modules - DEBUG: Print pathways \_Coverage 07/24/2020 01:03:41 AM - humann2.humann2 - INFO: TIMESTAMP: Completed computing pathways : 1 seconds 07/24/2020 01:03:41 AM - humann2.humann2 - INFO: Output files created: /home/bio/temp/AH1\_genefamilies.tsv /home/bio/temp/AH1\_pathabundance.tsv /home/bio/temp/AH1\_pathcoverage.tsv 07/24/2020 01:03:41 AM - humann2.utilities - DEBUG: Remove directory: /home/bio/temp/AH1\_humann2\_temp/tmpOXjg2v the above is the log when I tried to analysis my own data from swine after QC ,Could you tell me if it runs on the correct ways ---
**Author:** ![lauren.j.mciver](https://avatars.discourse-cdn.com/v4/letter/l/f05b48/32.png) [@lauren.j.mciver](https://forum.biobakery.org/u/lauren.j.mciver)\ **Post date:** [July 23, 2020, 6:21pm UTC](https://forum.biobakery.org/t/no-species-were-selected-from-the-prescreen-because-of-this-the-custom-chocophlan-database-is-empty-this-will-result-in-zero-species-specific-gene-families-and-pathways/765/2 "2020-07-23T18:21:16Z")
Hi Jamie, Thank you for the detailed post! It looks like there might be some formatting issues with a sequence in your fastq input file. This causes an error running MetaPhlAn (which runs the prescreen) and because of this error no species are identified so no custom nucleotide database is generated and so the nucleotide search portion of HUMAnN is not run. If you would double check your input file, more specifically the sequence that seems to have different lengths of bases vs quality values, resolving this formatting issue should resolve the error. I will copy the sequence error below for your reference which includes the sequence id. `ValueError: Lengths of sequence and quality values differs for A00920:121:HGNNYDRXX:2:2278:12391:36918:N:0:TACAAGTA+AGTCAACA#0/1 (250 and 1229240).` Thank you, Lauren ---
**Author:** ![khatso1](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/khatso1/32/1065_2.png) [@khatso1](https://forum.biobakery.org/u/khatso1)\ **Post date:** [October 6, 2021, 10:58am UTC](https://forum.biobakery.org/t/no-species-were-selected-from-the-prescreen-because-of-this-the-custom-chocophlan-database-is-empty-this-will-result-in-zero-species-specific-gene-families-and-pathways/765/3 "2021-10-06T10:58:55Z")
Hi Please assist, I have a similar problem will you assist, I ensured that I do not use the latest bowtie2. I got empty outputs please see the command and error below humann -i L001.fastq --protein-database uniref\_annotated --nucleotide-database chocophlan -o humann2\_results Output files will be written to: humann2\_results Removing spaces from identifiers in input file … Running metaphlan … Total species selected from prescreen: 0 Selected species explain 0.00% of predicted community composition No species were selected from the prescreen. Because of this the custom ChocoPhlAn database is empty. This will result in zero species-specific gene families and pathways. Running diamond … Aligning to reference database: uniref90\_demo\_prots\_v201901b.dmnd Running metaphlan … Total species selected from prescreen: 0 Selected species explain 0.00% of predicted community composition No species were selected from the prescreen. Because of this the custom ChocoPhlAn database is empty. This will result in zero species-specific gene families and pathways. ---
**Author:** ![franzosa](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/franzosa/32/3511_2.png) [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Post date:** [October 14, 2021, 8:23pm UTC](https://forum.biobakery.org/t/no-species-were-selected-from-the-prescreen-because-of-this-the-custom-chocophlan-database-is-empty-this-will-result-in-zero-species-specific-gene-families-and-pathways/765/4 "2021-10-14T20:23:54Z")
Are you sure that your FASTQ file is OK? This is saying that MetaPhlAn couldn’t identify anything from it. When this process completes, did you see any reads mapped to proteins (unclassified)? ---
**Author:** ![khatso1](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/khatso1/32/1065_2.png) [@khatso1](https://forum.biobakery.org/u/khatso1)\ **Post date:** [October 14, 2021, 9:36pm UTC](https://forum.biobakery.org/t/no-species-were-selected-from-the-prescreen-because-of-this-the-custom-chocophlan-database-is-empty-this-will-result-in-zero-species-specific-gene-families-and-pathways/765/5 "2021-10-14T21:36:18Z")
Dear Franzosa Thanks for tour response, I get empty output files. Regards ---
**Author:** ![khatso1](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/khatso1/32/1065_2.png) [@khatso1](https://forum.biobakery.org/u/khatso1)\ **Post date:** [October 22, 2021, 7:00pm UTC](https://forum.biobakery.org/t/no-species-were-selected-from-the-prescreen-because-of-this-the-custom-chocophlan-database-is-empty-this-will-result-in-zero-species-specific-gene-families-and-pathways/765/6 "2021-10-22T19:00:37Z")
Dear Franzosa There was a typo in my previous mail. I meant to say thank you for your prompt response. This is the first species of my fasta fiel > s\_\_Catonella\_morbi\_S001499241 > GATGAACGCTGGCGGCCTGCCTAACACATGCAAGTCGAACGGAGATTACGGACGGAAGTTTTCGGATGGAAGACTGTAATTCTTAGTGGCGGACGGGTGAGTAACGCGTGGGCAACCTGCCCTATACAGGGGGATAGCAGCTGGAAACGGCTGGTAAGACCGCATAAGTCGGCGAGACCGCATGGTTTTGCCGGGAAATGAGCAATCAGGTATAGGATGGGCCCGCGTCCGATTAGCCAGTTGGCAGGGTAAAAGCCTACCAAAGCAACGATCGGTAGCCGGACTGAGAGGTCGGACGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGGGTGAAGAAGTATTTCGGGATGTAAAGCTCTATCAGCAGGGAAGATGATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTTTGGCAAGTTGAGAGTGGAAGCAGGGGGCTCAACCCCCTGACTGCTCCCAAAACTGTTAAACTTGAGTATGGGAGAGGCAGGCGGAATTCCTAGTGTAGCGGTGAAATGCTTAGATATTAGGAAGAACACCGGTGGCGAAGGCGGCCTGCTGGACCAAAACTGACGCTGAGGCTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATACTAGGTGCTGGGAGGCATAAGCCTTTCGGTGCCGTCGCAAACGCATTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCGAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCCGGTCTTGAGATCCTCCTGAATACAGGGTAATGCCTGTAGTCCTTCGGGACAGGAGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTTTTAGTAGCCAGCGGTTCGGCCGGGCACTCTAGAAAGACTGCCGAGGATAACTCGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTACGACCGGGGCTACACACGTGCTACAATGGCAGTTACAGAGGGAAGCGAAGGCGTGAGCCGGAGCGAATCACAGAAAGGCTGCCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCT i get the error, please note that i have used metaphlan version 3.0.13, hoping for a prompt reply Running metaphlan … CRITICAL ERROR: Error executing: /apps/chpc/bio/anaconda3-2020.02/envs/Humann3/bin/metaphlan /mnt/lustre/users/kmasenya/RESDIRPharynx/test.fa -t rel\_ab -o /mnt/lustre/users/kmasenya/RESDIRPharynx/humann2\_results/test\_humann\_temp/test\_metaphlan\_bugs\_list.tsv --input\_type fasta --bowtie2out /mnt/lustre/users/kmasenya/RESDIRPharynx/humann2\_results/test\_humann\_temp/test\_metaphlan\_bowtie2.txt Error message returned from metaphlan : Traceback (most recent call last): File “/apps/chpc/bio/anaconda3-2020.02/envs/Humann3/bin/read\_fastx.py”, line 10, in sys.exit(main()) File “/apps/chpc/bio/anaconda3-2020.02/envs/Humann3/lib/python3.7/site-packages/metaphlan/utils/read\_fastx.py”, line 167, in main f\_nreads, f\_avg\_read\_length = read\_and\_write\_raw(f, opened=False, min\_len=min\_len, prefix\_id=prefix\_id) File “/apps/chpc/bio/anaconda3-2020.02/envs/Humann3/lib/python3.7/site-packages/metaphlan/utils/read\_fastx.py”, line 129, in read\_and\_write\_raw nreads, avg\_read\_length = read\_and\_write\_raw\_int(inf, min\_len=min\_len, prefix\_id=prefix\_id) File “/apps/chpc/bio/anaconda3-2020.02/envs/Humann3/lib/python3.7/site-packages/metaphlan/utils/read\_fastx.py”, line 116, in read\_and\_write\_raw\_int nreads = idx - discarded UnboundLocalError: local variable ‘idx’ referenced before assignment WARNING: It seems that you do not have Internet access. WARNING: Cannot connect to the database server. The latest available local database will be used. Regards