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=+= README =+============Last update: April 4, 2000 ============
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the latest version of this document can be found at:
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ftp://ftp.ncbi.nih.gov/fa2htgs/README
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After having consulted with NCBI staff (see contact information below)
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submitters from Genome Sequencing centers will establish what the best
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protocol will be for them to deposit their sequence submission data to
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One of these protocol may require the fa2htgs tool, present in this
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directory. fa2htgs is a program used to generate Seq-submits (an ASN.1
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sequence submission file) for high throughput genome sequencing
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projects. Presently we have built fa2htgs for the following platforms:
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win32/fa2htgs.exe (win95/NT)
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If fa2htgs is required for a platform not present here,
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please let us know (address below) and we will be happy to
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fa2htgs will read a FASTA file (or an Ace Contig file with Phrap sequence
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quality values), a Sequin submission template file, (to get contact
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and citation information for the submission), and a series of command line
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arguments (see below). This program will then combines these
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information to make a submission suitable for GenBank. Once you have
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generated your submission file, you need to follow the submission
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protocol (see the README present on your FTP account or mailed out to
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fa2htgs is intended for the automation by scripts for bulk submission of
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unannotated genome sequence. It can easily be extended from its current
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simple form to allow more complicated processing. A submission
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prepared with fa2htgs can also be read into Sequin, and then annotated
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more extensively. See the Sequin home page at:
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http://www.ncbi.nlm.nih.gov/Sequin/
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. Contacting NCBI about HTGS submissions and about using fa2htgs:
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Questions and concerns about this processing protocol, or how to
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use this tool should be forwarded to:
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htgs@ncbi.nlm.nih.gov.
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typing "fa2htgs -" will cause the program to show its command line
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arguments. Below we show these with additional comments (what we show
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within { } does not appear on the command line)
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fa2htgs 2.0 arguments:
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-i Filename for fasta input [File In]
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-t Filename for Seq-submit template [File In]
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default = template.sub
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-o Filename for asn.1 output [File Out] Optional
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-e Log errors to file named: [File Out] Optional
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-n Organism name? [String] Optional
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default = Homo sapiens
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-s Sequence name? [String]
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{ The sequence must have a name that is unique within }
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{ the genome center. We use the combination of the genome }
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{ center name (-g argument) and the sequence name (-s) to }
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{ track this sequence and to talk to you about it. }
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{ The name can have any form you like but must be unique }
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-l length of sequence in bp? [Integer]
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{ The length is checked against the actual number of }
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{ bases we get. For phase 1 and 2 sequence it is also }
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{ used to estimate gap lengths. For phase 1 and 2 }
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{ records, it is important to use a number GREATER than }
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{ the amount of provided nucleotide, otherwise this will }
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{ generate false 'gaps'. Here is assumed that the }
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{ putative full length of the BAC or cosmid will be used. }
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{ There should be at least 20 to 30 'n' in between the }
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{ segments (you can check for these in Sequin), as this }
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{ will ensure proper behavior when this sequence }
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{ is used with BLAST. Otherwise 'artifactual' unrelated }
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{ segment neighbors may be brought into proximity of }
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-g Genome Center tag? [String]
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{ This is probably the same as your login name on the }
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-p HTGS phase? [Integer]
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{ Phase 1 - a collection of unordered contigues with }
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{ gaps of unknown length. Phase 1 record must }
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{ at the very least have two segments with }
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{ Phase 2 - a series of ordered contigs, gap lengths may }
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{ be known. This could be a single sequence, }
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{ without gaps, if the sequence has ambiguities }
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{ which will be resolved. }
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{ Phase 3 - a single contiguous sequence. This sequenced }
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{ is finished, although it may, or may not }
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-a GenBank accession (if an update) [String] Optional
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{ this argument is required if this is an update, do }
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{ not use it if you are preparing a new submission }
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-r Remark for update? [String] Optional
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{ if this is an update, you can add a brief comment }
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{ (within "") describing the nature of the update }
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{ ("new sequence", "new citation", "updated features") }
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-c Clone name? [String] Optional
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{ will appear as /clone in the source feature }
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{ This could be the same as the -s argument (sequence }
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{ name) but this one will appear in the /clone qualifier }
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-h Chromosome? [String] Optional
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{ will appear as /chromsome in the source feature }
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-d Title for sequence? [String] Optional
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{ the text that will appear in the DEFINITION line }
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{ of the GenBank flatfile. }
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-m Take comment from template ? [T/F] Optional
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-u Take biosource from template ? [T/F] Optional
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-x Secondary accession number, separate by commas if multiple, s.t. U10000,L11000 [String] Optional
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[ ACCESSION AC000000 L00000 }
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{ | secondary accession number }
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{ primary accession number }
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{ In some cases a large segment will supercede another }
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{ or group of other accession numbers (records). These }
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{ records which are no longer wanted in GenBank should be }
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{ made secondary. Using the -x argument you can list the }
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{ Accession Numbers you want to make secondary. This will}
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{ instruct us to remove the accession number(s) from }
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{ GenBank, and will no longuer be part of the GenBank }
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{ release. They will nonetheless be available from Entrez.}
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{ !!GREAT CARE should be taken when using this argument!!!}
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{ inproper use of accession numbers here will result in }
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{ the innapropriate withdrawal of GenBank records from }
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{ GenBank, EMBL and DDBJ. We provide this parameter as }
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{ a conveniance to submitting centers, but this may need }
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{ removed if it is not used carefully. }
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-C Clone library name? [String] Optional
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{ will appear as /clone-lib="string" on the source feature }
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-M Map? [String] Optional
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{ will appear as /map="string" on the source feature }
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-O Filename for the comment: [File In] Optional
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{ will read the comment from a given file. }
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{ maximum 100 characters per line. }
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{ new lines can be incorporated with "~", and if you }
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{ actually want to include the "~" in your text, you }
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{ need to escape it with "`". Please ensure that the }
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{ correct format is obtained by viewing your comment }
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-T Filename for phrap input [File In] Optional
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{ Using this argument infers that you are NOT using the }
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-P Contigs to use, separate by commas if multiple [String] Optional
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{ if -P is not indicated with the -T option, then the }
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{ fragments will go in in the order that they are in the }
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{ ace file (which is appropriate for a phase 1 record, }
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{ but not for a phase 2 or 3. If you need to set the }
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{ order of the segments of the ace file, you need to set }
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{ it with the -P flag, like this: }
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{ -P "Contig1,Contig4,Contig3,Contig2,Contig5" }
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-A Filename for accession list input [File In] Optional
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{ Using this argument infers that you are NOT using the }
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{ -i or -T arguments above. The input file contains a }
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{ tab-delimited table with three to five columns, which }
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{ are accession number, start position, stop position, }
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{ and (optionally) length and strand. If start > stop, }
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{ the minus strand on the referenced accession is used. }
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{ A gap is indicated by the word "gap" instead of an }
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{ accession, 0 for the start and stop positions, and a }
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{ number for the length. }
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-X Coordinates are on the resulting sequence ? [T/F] Optional
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{ if -X is TRUE, then the coordinates in the input file }
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{ are on the resulting segmented sequence. This implies }
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{ that bases 1 through n of each accession are used. }
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{ if -X is FALSE, the coordinates are on the individual }
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{ accessions, and these need not start at base 1 of the }
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-D HTGS_DRAFT sequence? [T/F] Optional
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-S Strain name? [String] Optional
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-b Gap length [Integer]
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range from 0 to 1000000000
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-N Annotate assembly_fragments [T/F] Optional
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-6 SP6 clone (e.g., Contig1,left) [String] Optional
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-7 T6 clone (e.g., Contig2,right) [String] Optional
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-L Filename for phrap contig order [File In] Optional
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{ This is a tab-delimited file that can be used to drive }
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{ the order of contigs (normally specified by -P), as well }
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{ as indicating the SP6 and T7 ends. It can also be used }
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{ when contigs are known to be in opposite orientation. }
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{ Contig2 + 1 SP6 left }
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{ Contig1 - T7 right }
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{ The first column is the contig name, the second is the }
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{ orientation, the third is the fragment_group, the fourth }
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{ indicates the SP6 or T7 end, and the fifth says which }
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{ side of SP6 or T7 end had vector removed. }
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Presented here is an example of a phase 2 submission from an Arabidopsis
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sequencing center. It is followed by an command line arguments used in
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an example with a Phrap ace file.
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BEFORE YOU BEGIN: fa2htgs does depend on the presence of some external
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files. These are provided with Sequin, so if a networked version of
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Sequin is already installed (see URL above for Sequin info) all the
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default files that need to be present will be there and allow fa2htgs
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Here are the files you need (let's assume we have a 100Kb BAC):
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1) fasta file (example below)
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2) sequin submission file (more on this below)
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3) genome center name ("pgec" in this example, use your
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4) the sequence/clone name (this will *always* stay with the record)
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phase 1: multiple pieces, not in order (alway >= 2 pieces,
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phase 2: multiple pieces, in order, but can be as few as
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one unfinished sequence
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phase 3: 1 piece, where the sequence is "finished"
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6) the full sequence length, when the project is finished (eg 100000
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7) A new submission has no Accession Number, and and an update always
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does. You will need to keep track of this (ie which sequence name has
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which accession number)
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8) The organism, in this example "Arabidopsis thaliana"
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9) The chromosome number, 1 in this example.
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10) the output (file name) convention so far has been to call it the
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clone name.ss (eg P74A8.ss) "ss" is a seq-submit, or sequence
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submission. We then have our scripts/code report with the same file
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name convention. Also note that because we are working in Unix space,
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'case' of letter is important, and try to avoid 'metacharacters'
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so the phase 1 or 2 FASTA file will look like this (in this example,
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this is one has 3 segments, but you could (in phase 1) have many more):
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>P74A8 pcr product joining p130c12 and p91c10
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gatcagcccaaagcattgattaggggaacttacctgtagagggctgcagcaatggggaac
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acctggctgggtcacagagtggtcaatgcactccatgacttttgggtcaggacacagaaa
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gaaagagcggggaaccggggggccctacagtgatgaattatactaactgattttagaatg
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ttaaacaaacattgcatttccagaataaaccccatttagtaacgcatagtgtgcttgtat
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ctcagcctcccaaagtgctgggattatagacatgagccagcgcacctggctttgttagcc
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ttttcaaataactttttgaactttgttaattttttaattgcacgttttctccttcattta
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ctaattccattcaaaagtagcatcaatgagaataaattacttaggaatacatttaattaa
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aaagtgctagacttgtacactgaaaattacaaagtactctggagatatattc
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The first line has the seqence id, and a title, then each segment
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where you put a "?" if you don't know the distance between the pieces,
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or a number of bp if you do know the distance (eg 200 bp), and the
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other line is the fasta formated next segment (foobar). So that is it
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for phase 1 or 2. Phase 3 will be a single fasta file. All phase 1
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will probably always be >?.
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So the other thing you need is a submisssion prepared by sequin. This
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will allow you to put in the references, authors, Titles, submission
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information the way you want it. You simply need to make a 1 bp
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submission really. fa2htgs will read that file and copy the
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information over to the htgs information with the "real" data.
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So once you have made the submission, you deposit it on the FTP account
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under "SEQSUBMIT" directory, we have software that looks for it there
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every day, validate the center, clone (sequence) id's, check if it's an
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update and so on, and write a report that you can pick up the next
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It is good to put the output of fa2htgs in Sequin and validate the
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record. This is specially important for phase 3 records where many
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annotations may be present (added with the help of Sequin): Sequin has
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a very good validation suite (look under Search -> Validate)
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This finished record is now ready for deposition to your FTP account
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in the SEQSUBMIT directory.
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example of the command line arguments using quality score/Phrap ace file
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(all on tyhe same command line):
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./fa2htgs -t nuc1.sqn -o test.cmd32.out -s Phrap_Contig_Test2 -l 111505
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-g pgec -p 2 -h 1 -d Phrap_Contig_Test2 -n "Arabidopsis thaliana"
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-T g5129z079.ace -P "Contig1,Contig2,Contig4,Contig3,Contig7"
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example of a contig file for a yeast chromosome (with coordinates on the
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individual accessions):
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-- Questions about fa2htgs or how to submit?
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Just contact us at NCBI:
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e-mail: htgs@ncbi.nlm.nih.gov
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==============+= end of the fa2htgs README =+==========================
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doc/fa2htgs/README
