Genomics Application Notes

Minimal Cross Contamination In order to assess cross contamination of the libraries that might have occurred during construction, the scientists at the Center for Genomics and Bioinformatics used Blastn to search the reads against all three genomes, keeping the best hit from each genome and only considering matches that covered at least 35bp. Any read that mapped to the expected reference from the list of contaminants was rejected, as well as any read that mapped to both potential contaminants. The Biomek FX P added reagents starting in Column 1 and moving across the plate from left to right until it reached the end of the samples (Table 2). If sample carry-over during library construction occurred, we would expect to observe tomato sequences in the human UHR2-1 library prepared in well H5 of the library construction plate, but no cross contamination in the samples in column 1. Following sequencing, reads from the sequenced libraries were mapped against the correct reference genome and the reference genomes of possible contaminant species using BowTie with an edit distance of 3. Reads were only assigned to a particular species if the reads mapped only to a single genome. As can be seen in Table 5, the number of non-human reads in any of the human UHR libraries is quite low. The same holds true for the Drosophila libraries, in which we detect very few non- Drosophila reads. The high number of unassigned reads in the tomato, Tribolium, Enchenopa, and Umbonia libraries reflects the current state of reference genomes for these species, which are not as complete as those for human and Drosophila . Taken together, these results show that the libraries derived from the automated stranded mRNA method on the Biomek FXP show no discernible evidence of cross contamination during library construction. E7 Drosophila 19,214,090 0.000% 86.811% 0.026% 0.030% 0.016% 13.117% F7 Drosophila 24,124,996 0.000% 84.878% 0.021% 0.022% 0.000% 15.078% G7 Drosophila 23,859,338 0.000% 84.326% 0.020% 0.015% 0.000% 15.639% A8 Drosophila 24,089,160 0.000% 83.621% 0.088% 0.002% 0.041% 16.248% C11 Drosophila 25,074,808 0.000% 84.404% 0.014% 0.003% 0.033% 15.546% D11 Drosophila 20,772,508 0.000% 84.682% 0.012% 0.003% 0.027% 15.276% C8 Enchenopa 22,757,524 0.015% 0.109% 0.030% 0.008% 0.043% 99.796% H5 Human 18,216,660 0.008% 0.001% 81.131% 0.005% 0.012% 18.844% A6 Human 22,539,754 0.011% 0.006% 81.103% 0.003% 0.003% 18.875% H7 Human 9,140,720 0.008% 0.005% 81.833% 0.002% 0.020% 18.132% B8 Human 19,740,996 0.007% 0.002% 81.000% 0.002% 0.000% 18.989% E8 Human 23,576,296 0.010% 0.022% 80.714% 0.049% 0.023% 19.183% F10 Human 28,598,586 0.148% 0.009% 75.432% 0.052% 0.008% 24.351% G10 Human 28,372,488 0.024% 0.008% 80.398% 0.015% 0.003% 19.553% B11 Human 24,986,534 0.008% 0.003% 80.068% 0.004% 0.001% 19.915% E5 Tomato 29,987,210 0.090% 0.006% 0.019% 73.578% 0.011% 26.296% F5 Tomato 24,065,104 0.071% 0.008% 0.024% 76.216% 0.000% 23.680% G5 Tomato 27,038,110 0.068% 0.007% 0.019% 76.141% 0.000% 23.764% E11 Tomato 27,386,464 0.003% 0.019% 0.043% 48.448% 0.025% 51.463% F11 Tomato 26,794,430 0.281% 0.019% 0.031% 60.697% 0.004% 38.968% E10 Tribolium 29,664,318 0.025% 0.006% 0.062% 0.035% 74.705% 25.166% H10 Tribolium 20,463,272 0.056% 0.002% 0.028% 0.011% 69.281% 30.622% A11 Tribolium 26,114,234 0.009% 0.036% 0.021% 0.008% 73.260% 26.666% D8 Umbonia 25,890,506 0.027% 0.156% 0.011% 0.006% 0.045% 99.756% Table 5. Mapping to Reference Genomes LIBRARY PLATE WELL SPECIES TOTAL READS (ALL LANES, ALL READS) REFERENCE GENOME MAPPING PERCENTAGES Daphnia Drosophila H. sapiens Tomato Tribolium unassigned

B2013-14332 IB-18319A