Hello,
For my thesis, i have to do data analysis of my experiments. one of my experiments is the comparison of the r9.4.1 flow cell and the R10.4.1. flow cell. my mentor cannot help me for now which is why i am looking for help here. I have already written this data analysis but don't know if it is 100% correct. Anyone who can help me with this?
When the average read length is compared of both flow cells, shown in Figure 5, it can be observed that they are longer in the R9.4.1. flow cell. This is the case for all bacterial strains except S. suis ST2. For S. suis ST9, this cannot be compared due to a scarcity of material. Both the SQK-RBK004 kit (for the R9.4.1. flow cell) and the RBK-SQK114.24 kit (for the R10.4.1. flow cell) use transposase-based fragmentation. This involves fragmentation of DNA, after which the barcode is ligated to the fragment. Both kits are optimised for samples with fragments longer than 30 kb. For the SQK-RBK004 kit, the input is 400 ng, which means more gDNA is present and therefore more longer fragments. This compares with the SQK-RBK114.24 kit where the input is only 50 ng. As a result, more short fragments will be barcoded, resulting in a lower average read length. In both kits, there is a random distribution. As a result, the average read length may also be the same, as in the case of S. suis ST2. Thus, the result obtained corresponds to the expectation.
When comparing the average read quality of both flow cells, shown in Figure 6, it can be observed that for all bacterial strains, the read quality is better with the R10.4.1. flow cell. For S. suis ST9, this cannot be verified due to scarcity of material. When looking at the Phred quality scores (Q-scores), those of the SQK-RBK114.24 kit are higher than those of the RBK-SQK004 kit. The Phred quality score of the SQK-RBK114.24 kit can reach an accuracy of Q20+. The Phred quality score of the SQK-RBK004 kit can reach an accuracy of Q17. This Phred score is used to indicate quality in sequencing. Thus, the results obtained are in line with the expectation.
When the amount of reads are compared from both flow cells, shown in Figure 7, it can be observed that the amount of reads are higher in the R9.4.1. flow cell. Only for S. suis ST2 is this not the case. For S. suis ST9, this cannot be verified due to scarcity of material. The SQK-RBK004 kit requires an input of gDNA of 400 ng. This is higher than in the SQK-RBK114.24 kit, which requires an input of gDNA of 50 ng. The higher input at the SQK-RBK004 kit results in more reads. The translocation rate of the R9.4.1 flow cell (400 bp/s) is higher than that of the R10.4.1 flow cell (200 bp/s). The higher amount of gDNA and the faster translocation rate result in a higher amount of reads at the SQK-RBK004. The results obtained, except for S. suis ST2, are in line with expectations. A possible cause for this is the low concentration of S. suis ST2 obtained after extraction. Here, 400 ng of input could not be used due to a scarcity of material. Consequently, this may result in a lower amount of reads.
When the N50 values are compared from both flow cells, shown in Figure 8, it can be observed that the N50 values are higher in the R9.4.1 flow cell. Only for M. haemolytica 10531 is this not the case. For S. suis ST9, this cannot be verified due to scarcity of material. The N50 value represents the length of the shortest read in the group of the longest sequences which represents 50% of the nucleotides in the different sequences. Similar to the discussion in read length, the SQK-RBK004 kit requires more input (400 ng) than the SQK-RBK114.24 kit (50 ng). Consequently, more short fragments will be barcoded with the SQK-RBK114.24 kit, resulting in a lower average N50 value. Except for M. haemolytica 10531, the results obtained are in line with expectations. In both kits, there is a random distribution. This could be a possible cause that the N50 value is higher in the R10.4.1. flow cell.
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