Reference sequence statistics
For reports on results of read mapping, section two concerns the reference sequences. The reference identity part includes the following information:
- Reference name
- The name of the reference sequence.
- Reference Latin name
- The reference sequence's Latin name.
- Reference description
- Description of the reference.
) tab (learn more in Element information). Note that you need to create a new report if you want the information in the report to be updated. If you update the information for the reference sequence within the contig, you should know that it doesn't affect the original reference sequence saved in the Navigation Area.
The next part of the report reports coverage statistics including GC content of the reference sequence. Note that coverage is reported on two levels: including and excluding zero coverage regions. In some cases, you do not expect the whole reference to be covered, and only the coverage levels of the covered parts of the reference sequence are interesting. On the other hand, if you have sequenced the full genome that you use as reference, the overall coverage is probably the most relevant number (i.e. including zero coverage regions).
A position on the reference is counted as "covered" when at least one read is aligned to it. Note that unaligned ends (faded nucleotides at the ends) that are produced when mapping using local alignment do not contribute to the coverage. In the example shown in figure 19.14, there is a region of zero coverage in the middle and one time coverage on each side. Note that the gaps to the very right are within the same read which means that these two positions on the reference sequence are still counted as "covered".
Figure 19.3: A region of zero coverage in the middle and one time coverage on each side. Note that the gaps to the very right are within the same read which means that these two positions on the reference sequence are still counted as "covered".
The identity section is followed by some statistics on the zero-coverage regions; the number, minimum and maximum length, mean length, standard deviation, total length and a list of the regions. If there are too many regions, they will not all be listed in the report (if there are more than 20, only the first 10 are reported).
Next follow two bar plots showing the distribution of coverage with coverage level on the x-axis and number of positions with that coverage on the y-axis. An example is shown in figure 19.17.
Figure 19.4: Distribution of coverage - to the left for all the coverage levels, and to the right for coverage levels within 3 standard deviations from the mean.
The graph to the left shows all the coverage levels, whereas the graph to the right shows coverage levels within 3 standard deviations from the mean. The reason for this is that for complex genomes, you will often have a few regions with extremely high coverage which will affect the resolution of the graph, making it impossible to see the coverage distribution for the majority of the references. These coverage outliers are excluded when only showing coverage within 3 standard deviations from the mean. Note that zero-coverage regions are not shown in the graph but reported in text below (this information is also in the zero-coverage section). Below the second coverage graph there are some statistics on the data that is outside the 3 standard deviations.
One of the biases seen in sequencing data concerns GC content. Often there is a correlation between GC content and coverage. In order to investigate this correlation, the report includes a graph plotting coverage against GC content (see figure 19.16). Note that you can see the GC content for each reference sequence in the table above.
Figure 19.5: The plot displays, for each GC content level (0-100 %), the mean read coverage of 100bp reference segments with that GC content.
The plot displays, for each GC content level (0-100 %), the mean read coverage of 100bp reference segments with that GC content.
At the end follows statistics about the reads which are the same for both reference and de novo assembly (see Read statistics).
Next follow two bar plots showing the distribution of coverage with coverage level on the x-axis and number of positions with that coverage on the y-axis. An example is shown in figure 19.17.
Figure 19.6: Distribution of coverage - to the left for all the coverage levels, and to the right for coverage levels within 3 standard deviations from the mean.
The graph to the left shows all the coverage levels, whereas the graph to the right shows coverage levels within 3 standard deviations from the mean. The reason for this is that for complex genomes, you will often have a few regions with extremely high coverage which will affect the resolution of the graph, making it impossible to see the coverage distribution for the majority of the references. These coverage outliers are excluded when only showing coverage within 3 standard deviations from the mean. Below the second coverage graph there are some statistics on the data that is outside the 3 standard deviations. At the end follows statistics about the reads which are the same for both reference and de novo assembly (see Read statistics).