Bias in Ligation-Based Small RNA Sequencing Library Construction Is Determined by Adaptor and RNA Structure
Fig 8
HTS results from libraries made using different oligonucleotide mixtures.
Three oligonucleotide mixtures were subjected to HTS using different adaptors, as indicated. The adaptors used in library construction are represented by solid lines (defined sequence), jagged lines (randomized sequence), and a solid line with a gray background (region that is complementary to the 5’-end of the 3’ adaptor) and their sequences can be found in S1 Table. These oligonucleotide mixtures each contained the same 50 sequences, but the sequences were mixed in different amounts; either equivalent or different 500-fold spreads in concentration (S6 Table). Normalized reads for the 50 miRNA sequences in each data set are presented as individual data points on a logarithmic scale. Each miRNA’s normalized reads value was divided by its expected value given the concentration of the miRNA in the defined mix. A result of “1” after this division indicates that the normalized reads were equal to the expected value (dark gray line). The interval of 2-fold from the expected value (equivalent to a 2-fold over or under representation) is shown with light gray lines and 10-fold under the expected value is shown with a red line. The percentage of data points that are >10-fold under the expected value is shown next to a vertical red bar, and the percentage <2-fold from the expected value is shown next to a vertical green bar. The percentages of data points in other regions are shown next to vertical yellow bars. The Mann-Whitney test was used to compare each IT MidRand data set to its corresponding A1 adaptor data set to determine the statistical significance of the difference between the two sets. Two-tailed p values are indicated as (****) p < 0.0001. The values used to make the graph can be found in S6 Table.