## Optimization of the graph using paired reads

When paired reads are available, we can use the paired information to resolve large repeat regions that are not spanned by individual reads, but are spanned by read pairs. Given a set of paired reads that align to two nodes connected by a repeat region, the repeat region may be resolved for those nodes if we can find a path connecting the nodes with a length corresponding to the paired read distance. However, such a path must be supported by a minimum of four sets of paired reads before the repeat is resolved.

If it's not possible to resolve the repeat, scaffolding is performed where paired read information is used to determine the distances between contigs and the orientation of these. Scaffolding is only considered between contigs with a minimum length of 120 to ensure that enough paired read information is available. An iterative greedy approach is used when performing scaffolding where short gaps are closed first, thus increasing the paired read information available for closing gaps (see figure 28.12).

Figure 28.12: Performing iterative scaffolding of the shortest gaps allows long pairs to be optimally used. i1 shows three contigs with dashed arches indicating potential scaffolding. i2 is after first iteration when the shortest gap has been closed and long potential scaffolding has been updated. i3 is the final results with three contigs in one scaffold.

Contigs in the same scaffold are output as one large contig with Ns inserted in between. The number of Ns inserted correspond to the estimated distance between contigs, which is calculated based on the paired read information. More precisely, for each set of paired reads spanning two contigs a distance estimate is calculated based on the supplied distance between the reads. The average of these distances is then used as the final distance estimate. The distance estimate will often be negative which happens when the paired information indicate that two contigs overlap. The assembler will attempt to align the ends of such contigs and if a high quality overlap is found the contigs are joined into a single contig. If no overlap is found, the distance estimate is set to two so that all remaining scaffolds have positive distance estimates.

Please note that Ns can also be present in output scaffolds, because input sequencing reads themselves contain Ns. Furthermore, there was an issue in CLC Genomics Workbench 6.0.1, Genomics Server 5.0.1, Assembly Cell 4.0.2 and all earlier versions of these products. In these versions parts of the scaffolds, which were build by using paired reads spanning a region (this part of the scaffold is in the middle of two reads) and those covering a region (there is a sequence for the part of the scaffold), also included Ns. In newer versions, the corresponding sequence from the alignment of reads covering the region is used in the scaffolds instead of Ns.

Additional information about repeats being resolved using paired reads and scaffolded contigs is available as annotations on the contig sequences and as summary in the report (see De novo assembly report). This information can also be exported in AGP format.

The annotations in table format can be viewed by clicking the "Show Annotation Table" icon () at the bottom of the viewing area. "Show annotation types" in the side panel allows you to select the annotation "Scaffold" among a list of other annotations. The annotations tell you about the scaffolding that was performed by the de novo assembler. That is, it tells you where particular contigs, those areas containing complete sequence information, were joined together across regions without complete sequence information. For the GFF format there are three types of annotations:

• Scaffold refers to the estimated gap region between two contigs where Ns are inserted.
• Contigs joined refers to the join of two contigs connected by a repeat or another ambiguous structure in the graph, which was resolved using paired reads. Can also refer to overlapping contigs in a scaffold that were joined using an overlap.
• Alternatives excluded refers to the exclusion of a region in the graph using paired reads, which resulted in a join of two contigs.