hepcat72
commented
9 years ago Hi Koji,
Thanks for using CFF. Let me explain how getReals.pl works, but first, let me back up to the previous step....
getCandidates.pl selects sequences it thinks are real by comparing each sequence with the abundance you would expect to get if it was an erroneous sequence (as compared to neighboring sequences that differ only by 1 base). It uses the estimated error rate of each nucleotide change to predict the likelihood that its neighbors were misread during PCR to produce it. If the sequence's actual abundance is above the erroneous predicted abundance by a given threshold, it is assumed to be real.
Now getReals.pl takes it a step further. getReals.pl looks across multiple samples (e.g. a time series) and considers all the candidates from different sample files and if it sees the same candidate sequence a threshold number of times, it assumes the sequence is real. It also does filtering for chimeras.
So, running getReals.pl requires multiple sample files (at least 2) in order to run. Essentially, your results with a single sample file are the candidate files.
If you wish to perform chimera filtering on a single sample file, currently, you would have to run uchime on your own. getReals.pl runs ucime in 2 separate calls:
usearch -usearch_global $tmp_lib -db $gcand_file -id 1.0 -idprefix $seqlen -matched $cands_with_global_abund -strand plus -quiet
usearch -uchime_denovo $cands_with_global_abund -minuniquesize 2 -nonchimeras $tmp_out_file $aln_arg -quiet
where:
$tmp_lib = a temporary library file output in the first step. $gcand_file = your candidates file. $seqlen = the length of your sequences. $cands_with_global_abund = a temporary candidate file output in the first step. $tmp_out_file = your chimera output file. $aln_arg = your chimera alignment output file.
Good luck, Rob
Robert William Leach 133A Carl C. Icahn Lab Lewis-Sigler Institute for Integrative Genomics Princeton University Princeton, NJ 08544
On Jun 16, 2015, at 7:05 AM, kojiyasuda notifications@github.com wrote:
It looks like my run was terminated at "getReals" step and I am not sure how to fix this. Does this error make sense to you? I placed my input fna file containing 16S V4, Illumina stitched 250nt amplicons into Caporaso_FSTA folder(after removing the original examples) since the example1 ran nicely and I didn't know how to run a new command. Hence it says Caporaso_FASTA, but it actually contained my fna file that I renamed to one of the example fna files. Thanks for looking into this! Koji
/Users/kojiyasuda/Desktop/CFF-master/samples/run_example1.tcsh
RUNNING run_CFF_on_FastA.tcsh
Start time: Mon Jun 15 18:57:44 EDT 2015 Trim length: 130 Z-score threshold: 2 Magnitude over N0 Threshold: 10 Nominations threshold: 2 OUTPUT DIRECTORY: Caporaso_FASTA_out mergeSeqs.pl 'Caporaso_FASTA/L6S2?_19???.fna' -f 'global_library.fna' --outdir 'Caporaso_FASTA_out/2_lib' -o .lib -b 130 -p '' -- 1989 seconds neighbors.pl 'Caporaso_FASTA_out/1_lib/global_library.fna' -o .nbrs -- 995 seconds errorRates.pl 'Caporaso_FASTA_out/1_lib/global_library.fna' -n 'Caporaso_FASTA_out/1_lib/global_library.fna.nbrs' -z 2 -o .erates -- 262 seconds nZeros.pl 'Caporaso_FASTA_out/1_lib/{L6S2?_19???.fna}.lib' -n 'Caporaso_FASTA_out/1_lib/global_library.fna.nbrs' -r 'Caporaso_FASTA_out/1_lib/global_library.fna.erates' -o .n0s --outdir 'Caporaso_FASTA_out/2_n0s' -- 348 seconds getCandidates.pl 'Caporaso_FASTA_out/2_n0s/{L6S2?_19???.fna}.lib.n0s' -o .cands -h 10 --outdir 'Caporaso_FASTA_out/3_cands' -- 256 seconds getReals.pl -i 'Caporaso_FASTA_out/3_cands/{L6S2?_19???.fna}.lib.n0s.cands' -n 'Caporaso_FASTA_out/2_n0s/{L6S2?_19???.fna}.lib.n0s' -f 'Caporaso_FASTA_out/1_lib/global_library.fna' -k 2 --outdir 'Caporaso_FASTA_out/4_reals_table'ERROR1: Too few candidates files (-i) supplied. The number of minimum candidacies (-k): [2] requires at least as many files supplied to each of the -i and -n (backwards-compatible with -d) options. I.e. -i requires at least [2] files and -n requires at least [2] files. If you only have 1 of each file, then this script should not be applied unless you set -k to 1, which will allow you to filter for chimeras at least.
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kojiyasuda
It looks like my run was terminated at "getReals" step and I am not sure how to fix this. Does this error make sense to you? I placed my input fna file containing 16S V4, Illumina stitched 250nt amplicons into Caporaso_FSTA folder(after removing the original examples) since the example1 ran nicely and I didn't know how to run a new command. Hence it says Caporaso_FASTA, but it actually contained my fna file that I renamed to one of the example fna files. Thanks for looking into this! Koji
/Users/kojiyasuda/Desktop/CFF-master/samples/run_example1.tcsh
RUNNING run_CFF_on_FastA.tcsh
Start time: Mon Jun 15 18:57:44 EDT 2015 Trim length: 130 Z-score threshold: 2 Magnitude over N0 Threshold: 10 Nominations threshold: 2 OUTPUT DIRECTORY: Caporaso_FASTA_out mergeSeqs.pl 'Caporaso_FASTA/L6S2?_19???.fna' -f 'global_library.fna' --outdir 'Caporaso_FASTA_out/2_lib' -o .lib -b 130 -p '' -- 1989 seconds neighbors.pl 'Caporaso_FASTA_out/1_lib/global_library.fna' -o .nbrs -- 995 seconds errorRates.pl 'Caporaso_FASTA_out/1_lib/global_library.fna' -n 'Caporaso_FASTA_out/1_lib/global_library.fna.nbrs' -z 2 -o .erates -- 262 seconds nZeros.pl 'Caporaso_FASTA_out/1_lib/{L6S2?_19???.fna}.lib' -n 'Caporaso_FASTA_out/1_lib/global_library.fna.nbrs' -r 'Caporaso_FASTA_out/1_lib/global_library.fna.erates' -o .n0s --outdir 'Caporaso_FASTA_out/2_n0s' -- 348 seconds getCandidates.pl 'Caporaso_FASTA_out/2_n0s/{L6S2?_19???.fna}.lib.n0s' -o .cands -h 10 --outdir 'Caporaso_FASTA_out/3_cands' -- 256 seconds getReals.pl -i 'Caporaso_FASTA_out/3_cands/{L6S2?_19???.fna}.lib.n0s.cands' -n 'Caporaso_FASTA_out/2_n0s/{L6S2?_19???.fna}.lib.n0s' -f 'Caporaso_FASTA_out/1_lib/global_library.fna' -k 2 --outdir 'Caporaso_FASTA_out/4_reals_table'ERROR1: Too few candidates files (-i) supplied. The number of minimum candidacies (-k): [2] requires at least as many files supplied to each of the -i and -n (backwards-compatible with -d) options. I.e. -i requires at least [2] files and -n requires at least [2] files. If you only have 1 of each file, then this script should not be applied unless you set -k to 1, which will allow you to filter for chimeras at least.