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Include consensus – If this check box is selected, the application will generate all the output files, including the output files related to the generation of contigs and scaffold information. If it is not selected, the application will perform the assembly and will output files (454ReadStatus.txt and 454TrimStatus.txt) associated with the creation of the multiple-alignments, but will not output files (454AllContigs.fna and .qual, 454LargeContigs.fna and .qual, scaffold files, etc.) or metrics (latter parts of 454NewblerMetrics.txt) involved with contig or consensus information. The file ContigGraph.txt is output, but is empty. If this option is unchecked, the assembly will run faster, providing an approximate idea of the quality of the data, based on the percentage of reads assembled.

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The Reads limited to one contig option tells the assembler to generate output where each read occurs in one and only one contig (i.e. read alignments are not allowed to span across multiple contigs). This creates output ace and consed files suitable for 3^rd party software for further data analysis. The function operates after the contigs and scaffolds are created, by attempting to remove reads from the ends of contigs that contain sets of reads that branch to multiple contigs. When a read is found that extends from such a contig to another contig end that doesn’t branch, the part of the read in the branched contig is removed and placed at the end of the part of the read in the non-branching contig.

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The Quick output option generates output faster than using the default setting by disabling the “Computing signals…” computation. The default behavior of the assembler requires that all reads be re-read so that signal and quality information can be used to compute consensus (see Section 1.1). If this option is selected, the accuracy of the consensus may be degraded (i.e., more consensus errors may be generated) as the distribution statistics are not generated to assist in the basecalling step.

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Output trimmed reads – select this option to output the read sequences to a .fna file after trimming has been performed (includes vector / primer / Paired End linker / MIDs / low quality trimming). If available, the quality value for the output reads will be placed in a .qual file.

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Output scaffolds – select this option to output scaffoldContigs to a .fna file and associated .qual file. When ‑scaffold is selected, the contents of 454Scaffolds.txt will represent scaffoldContigs and gaps rather than the contigs found in the allContigs file. An additional file, 454ContigScaffolds.txt, will be produced that is identical to the 454Scaffolds.txt file produced without the –scaffold option. The scaffoldContig names found in the 454Scaffolds.txt file represent the scaffoldContigs found in the 454ScaffoldContigs.fna and .qual files.

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The ACE Format option determines whether or what form of ACE file(s) are output by the application. The default is “Single ACE file for small genomes”. See section 1.15.1.5 for a description of the 454Contigs.ace file and of the ace and consed directories. By default, files consistent with version 17.0 or higher of consed are generated.

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The Alignment Info option has 3 possible settings.

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Output – turns on generation of the 454AlignmentInfo.tsv file

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Output small – the file will be generated unless there are more than 4 M reads or the assembly generates contigs with a total length in excess of 40 Mbp.

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Pairwise Alignment – Determines whether the 454PairAlign.txt file is output; this file contains the overlaps used by the assembler.

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Default is “None”. See section 1.15.1.8 for a description of the 454PairAlign.txt file.

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Ace read mode – When the ACE file is generated, output reads using either the raw, complete basecalled read or using just the trimmed portion of the reads (after low quality, vector and key trimming)

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All contig threshold – The minimum number of bases for a contig to be output in the 454AllContigs.fna file

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Large contig threshold – The minimum number of bases for a contig to be output in the 454LargeContigs.fna file

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