.\" Man page generated from reStructuredText. . .TH "ECOPCR" "1" "Jan 21, 2019" " 1.02 12" "OBITools" .SH NAME ecoPCR \- description of ecoPCR . .nr rst2man-indent-level 0 . .de1 rstReportMargin \\$1 \\n[an-margin] level \\n[rst2man-indent-level] level margin: \\n[rst2man-indent\\n[rst2man-indent-level]] - \\n[rst2man-indent0] \\n[rst2man-indent1] \\n[rst2man-indent2] .. .de1 INDENT .\" .rstReportMargin pre: . RS \\$1 . nr rst2man-indent\\n[rst2man-indent-level] \\n[an-margin] . nr rst2man-indent-level +1 .\" .rstReportMargin post: .. .de UNINDENT . RE .\" indent \\n[an-margin] .\" old: \\n[rst2man-indent\\n[rst2man-indent-level]] .nr rst2man-indent-level -1 .\" new: \\n[rst2man-indent\\n[rst2man-indent-level]] .in \\n[rst2man-indent\\n[rst2man-indent-level]]u .. .sp \fBecoPCR\fP \fIin silico\fP PCR preserves the taxonomic information of the selected sequences, and allows various specified conditions for the \fIin silico\fP amplification. .sp Additionally to the different options, the command requires two arguments corresponding to the two primers. .SH REFERENCES .INDENT 0.0 .INDENT 3.5 Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H (2010) ITS as an environmental DNA barcode for fungi: an \fIin silico\fP approach reveals potential PCR biases BMC Microbiology, 10, 189. .sp Ficetola GF, Coissac E, Zundel S, Riaz T, Shehzad W, Bessiere J, Taberlet P, Pompanon F (2010) An \fIin silico\fP approach for the evaluation of DNA barcodes. BMC Genomics, 11, 434. .UNINDENT .UNINDENT .SH ECOPCR SPECIFIC OPTIONS .INDENT 0.0 .INDENT 3.5 .INDENT 0.0 .TP .B \-d Filename containing the database used for the \fIin silico\fP PCR. The database must be in the \fBecoPCR format\fP (see obiconvert). .sp \fBWARNING:\fP .INDENT 7.0 .INDENT 3.5 This option is compulsory. .UNINDENT .UNINDENT .UNINDENT .INDENT 0.0 .TP .B \-e Maximum number of errors (mismatches) allowed per primer (default: 0). See example 2 for avoiding errors on the 3’ end of the primers. .UNINDENT .INDENT 0.0 .TP .B \-l Minimum length of the \fIin silico\fP amplified DNA fragment, excluding primers. .UNINDENT .INDENT 0.0 .TP .B \-L Maximum length of the \fIin silico\fP amplified DNA fragment, excluding primers. .UNINDENT .INDENT 0.0 .TP .B \-r Only the sequence records corresponding to the taxonomic group identified by its \fBTAXID\fP are considered for the \fIin silico\fP PCR. The \fBTAXID\fP is an integer that can be found either in the NCBI taxonomic database, or using the ecofind program. .UNINDENT .INDENT 0.0 .TP .B \-i The sequences of the taxonomic group identified by its \fBTAXID\fP are not considered for the \fIin silico\fP PCR. .UNINDENT .INDENT 0.0 .TP .B \-c Considers that the sequences of the database are circular (e.g. mitochondrial or chloroplast DNA). .UNINDENT .INDENT 0.0 .TP .B \-D Keeps the specified number of nucleotides on each side of the \fIin silico\fP amplified sequences, (including the amplified DNA fragment plus the two target sequences of the primers). .UNINDENT .INDENT 0.0 .TP .B \-k Print in the programme output the kingdom of the \fIin silico\fP amplified sequences (default: print the superkingdom). .UNINDENT .INDENT 0.0 .TP .B \-m <1|2> Defines the method used for estimating the Tm (melting temperature) between the primers and their corresponding target sequences (default: 1). .INDENT 7.0 .INDENT 3.5 1 SantaLucia method (SantaLucia J (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest\-neighbor thermodynamics. PNAS, 95, 1460\-1465). .sp 2 Owczarzy method (Owczarzy R, Vallone PM, Gallo FJ \fIet al.\fP (1997) Predicting sequence\-dependent melting stability of short duplex DNA oligomers. Biopolymers, 44, 217\-239). .UNINDENT .UNINDENT .UNINDENT .INDENT 0.0 .TP .B \-a Salt concentration used for estimating the \fITm\fP (default: 0.05). .UNINDENT .INDENT 0.0 .TP .B \-h Print help. .UNINDENT .UNINDENT .UNINDENT .SH OUTPUT FILE .INDENT 0.0 .INDENT 3.5 The output file contains several columns, with ‘|’ as separator, and describes the properties of the \fIin silico\fP amplified sequences. .sp column 1: sequence identification in the reference database (= accession number when using EMBL or GenBank for building the reference database) .sp column 2: length of the original sequence .sp column 3: scientific name as indicated in the reference database .sp column 4: taxonomic rank as indicated in the reference database .sp column 5: \fItaxid\fP of the species .sp column 6: scientific name of the species .sp column 7: \fItaxid\fP of the genus .sp column 8: genus name .sp column 9: \fItaxid\fP of the family .sp column 10: family name .sp column 11: \fItaxid\fP of the super kingdom (or of the kingdom if the \fB\-k\fP option is set) .sp column 12: super kingdom name (or kingdom name if the \fB\-k\fP option is set) .sp column 13: strand (D or R, corresponding to direct or reverse, respectively) .sp column 14: target sequence of the first primer .sp column 15: number of mismatches for the first primer .sp column 16: target sequence of the second primer .sp column 17: number of mismatches for the second primer .sp column 18: length of the amplified fragment (excluding primers) .sp column 19: sequence .sp column 20: definition .UNINDENT .UNINDENT .SH EXAMPLES .INDENT 0.0 .INDENT 3.5 \fIExample 1:\fP .INDENT 0.0 .INDENT 3.5 .INDENT 0.0 .INDENT 3.5 .sp .nf .ft C > ecoPCR \-d mydatabase \-e 3 \-l 50 \-L 500 \e TCACAGACCTGTTATTGC TYTGTCTGSTTRATTSCG > mysequences.ecopcr .ft P .fi .UNINDENT .UNINDENT .sp Launches an \fIin silico\fP PCR on mydatabase (see obiconvert for a description of the database format), with a maximum of three mismatches for each primer. The minimum and maximum amplified sequence lengths (excluding primers) are 50 bp and 500 bp, respectively. The primers used are TCACAGACCTGTTATTGC and TYTGTCTGSTTRATTSCG (possibility to use IUPAC codes). They amplify a short portion of the nuclear 18S gene. The results are saved in the \fImysequence.ecopcr\fP file. .UNINDENT .UNINDENT .sp \fIExample 2:\fP .INDENT 0.0 .INDENT 3.5 .INDENT 0.0 .INDENT 3.5 .INDENT 0.0 .INDENT 3.5 .sp .nf .ft C > ecoPCR \-d mydatabase \-e 2 \-l 80 \-L 120 \-D 50 \-r 7742 \e TTAGATACCCCACTATG#C# TAGAACAGGCTCCTCTA#G# > mysequences.ecopcr .ft P .fi .UNINDENT .UNINDENT .UNINDENT .UNINDENT .sp Launches an \fIin silico\fP PCR on mydatabase (see obiconvert for a description of the database format), with a maximum of two mismatches for each primer, but with a perfect match on the last two nucleotides of the 3’ end of each primer (a perfect match can be enforced by adding a ‘#’ after the considered nucleotide). The minimum and maximum amplified sequence lengths (excluding primers) are 80 bp and 120 bp, respectively. The \fB\-D\fP option keeps 50 nucleotides on each side of the \fIin silico\fP amplified sequences, (including the amplified DNA fragment plus the two target sequences of the primers). The primers used are TTAGATACCCCACTATGC and TAGAACAGGCTCCTCTAG. They amplify a short portion of the mitochondrial 12S gene. The \fB\-r\fP option restricts the search to vertebrates (7742 is the taxid of vertebrates). The results are saved in the \fBmysequence.ecopcr\fP file. .UNINDENT .UNINDENT .UNINDENT .UNINDENT .SH ECOPCR USED SEQUENCE ATTRIBUTES .INDENT 0.0 .INDENT 3.5 .INDENT 0.0 .IP \(bu 2 taxid .UNINDENT .UNINDENT .UNINDENT .SH AUTHOR The OBITools Development Team - LECA .SH COPYRIGHT 2019 - 2015, OBITool Development Team .\" Generated by docutils manpage writer. .