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1. GS De Novo Assembler : 1.15 GS De Novo Assembler Output : 1.15.2 GS De Novo Assembler cDNA / Transcriptome Output
1.15.2
GS De Novo Assembler cDNA / Transcriptome Output
1.15.2.1
454Isotigs.fna, 454Isotigs.qual, and 454Isotigs.txt files
The files 454LargeContigs.fna and 454LargeContigs.qual (1.15.1.2) are not generated for a cDNA / transcriptome assembly project. However, the new files 454Isotigs.fna (Figure 35) and 454Isotigs.qual files are generated (in addition to the 454AllContigs.fna and 454AllContigs.qual files). Similar to contig files, they contain respectively FASTA sequences and their quality scores. These files contain the isotig sequences traversed from the multiple-alignment graph structure (i.e. from the isogroup), but they also can contain single contigs which haven’t been traversed (and therefore aren’t considered isotigs) because of thresholds, limits, cycles, etc.
Figure 35: Portion example of the 454Isotigs.fna file for a cDNA assembly project
 
Also, a special file, 454Isotigs.txt is generated with content and format identical to the 454Scaffolds.txt file(Figure 36).
Figure 36: Portion example of the 454Isotigs.txt file for a cDNA assembly project
 
1.15.2.2
454Isotigs.faa and 454IsotigOrfAlign.txt
The 454Isotigs.faa file is a protein sequence FASTA file for all ORFs found in the given isotig nucleotide sequence, that are encoded by 100 or more nucleotides (Figure 37).
The amino acid sequences are sorted by length (for each isotig), and are preceded by a description line that consists of following tab-delimited information:
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Isotig name (as it appears in all other output files)
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Isotig nucleotide start position (1-based, inclusive)
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Isotig nucleotide end position (1-based, inclusive)
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ORF frame {-3, -2, -1, +1, +2, +3}
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Nucleotide sequence length (including stop codon, if present in the sequence)
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Protein sequence length (excluding stop codon, if present in the sequence)
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Number of M (Methionine) codons
 
The data in the 454isotigs.faa file is based on the assumption that the isotig represents a complete, full-length transcript. Multiple open reading frames may be reported, but only one is correct. The longest ORF is most likely the correct one, but this must be verified by the user. The application does not evaluate the likelihood that the ORF is correct, for example by evaluating codon usage.
 
Figure 37: Portion example of the 454Isotigs.faa file for a cDNA assembly project
The 454IsotigOrfAlign.txt file contains one or more lines for isotig nucleotide sequence, and each amino acid sequence encoded by a particular ORF found in the isotig (Figure 38). The isotig sequence is always first and amino acid sequences are sorted by start position in the isotig (or by end position for negative frames). Each line consists of following items:
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Accno: The isotig name or ORF info (frame:startBase..endbase); the longest ORF for each isotig ends with an asterisk “*”
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First position of the current section: nucleotide base position for an isotig, or amino acid position for an ORF sequence; a negative frame indicates it is greater than the last position for the current section
•
Sequence: Section of nucleotide or amino acid sequence
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Last position of the current section: Nucleotide base position for isotig, or amino acid position for an ORF sequence; a negative frame indicates it is less than the first position for the current section
Figure 38: Portion example of the 454IsotigOrfAlign.txt file for a cDNA assembly project
 
1.15.2.3
454Isotigs.ace and consed/…
The cDNA assembler produces an ACE file similar to that produced by the genomic assembler (see 1.15.1.5). Isotigs and large contigs that do not appear in any isotig are represented in the ACE file along with reads used to construct their multiple alignments.
 
1.15.2.4
454NewblerMetrics.txt
The consensusResults group of the 454NewblerMetrics file for cDNA projects displays output similar but not identical to those output by genomic projects in that it gives metrics for isogroups and isotigs in addition to the metrics for the large contigs and all contigs (Figure 39).
Figure 39: 454NewblerMetrics file, consensusResults group example for a cDNA project
 
1.15.2.5
454RefLink.txt
Another file specific to cDNA / transcriptome projects, 454RefLink.txt, is generated with a format like UCSC’s refLink.txt files. This can be used as an annotation file for further mapping projects of the cDNA / transcriptome assembly products (isotigs and contigs).
Figure 40: 454RefLink.txt file example
 
1.15.2.6
454IsotigsLayout.txt
Finally, the file 454IsotigsLayout.txt gives a visual representation of how the contigs are laid along each isotig in the isogroup:
Figure 41: Partial view of upper portion of the 454IsotigsLayout.txt file
where ">>>>>" and "<<<<<" denote whether the particular contig was traversed from the 5' to the 3’ end or vice -versa.
The lengths given in the top row are contig lengths, and the lengths on the right-most column are isotig lengths.
In addition, at the end of this file there are 5 histograms (Figure 42):
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counts of isogroup contigs
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counts of isogroup isotigs
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counts of isotig contigs
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lengths of contigs (binned into 100bp bins)
•
lengths of isotigs (binned into 100bp bins)
 
A
 
B
 
C
 
D
 
E
Figure 42: Partial view of lower portion of the 454IsotigsLayout.txt file
At the very end of the 454IsotigsLayout.txt file is information about how many regular isotigs are generated and how many non-traversed contigs remain. The status column gives the reason each contig was not traversed (thresholds, limits, cycles, etc.). For some contigs, the status can be "none", meaning they weren't reached at all because the traversal stopped before.
Figure 43: The end of the 454IsotigsLayout.txt file