Genomics Application Notes

Table 2. RNA Template Sample Setup on a 96 Well Plate

SPECIES TYPE Row/ Column 1

2 3 4 5

6

7

8

9

10

11

12

A Tomato Tomato Tomato Tomato Tomato Human UHR D. melanogaster D. melanogaster D. pulex Tribolium Tribolium Tomato B Tomato Tomato Tomato Tomato Tomato D. melanogaster D. melanogaster Human UHR D. pulex Tribolium Human UHR Tomato

Enchenopa binotata Umbonia crassicornis

C

Tomato Tomato Tomato Tomato Tomato D. melanogaster D. melanogaster

D. pulex Tribolium D. melanogaster Tomato

Human UHR

D

Tomato Tomato Tomato Tomato Tomato D. melanogaster D. melanogaster

Tribolium D. melanogaster Tomato

E Tomato Tomato Tomato Tomato Tomato D. melanogaster D. melanogaster Human UHR D. pulex Tribolium Tomato Tomato

Human UHR Human UHR

F Tomato Tomato Tomato Tomato Tomato D. melanogaster D. melanogaster

D. pulex

Tribolium

Tomato Tomato

G

Tomato Tomato Tomato Tomato Tomato D. melanogaster D. melanogaster

D. pulex

Tribolium

Tomato Tomato

Human UHR

H

Tomato Tomato Tomato Tomato

D. melanogaster Human UHR

D. pulex

Tribolium Tribolium Tomato Tomato

Consistent Library Results Total RNA from each sample shown in Table 2 was re- suspended in a final volume of 50 μ L prior to starting the method. RNA fragmentation time was 7 minutes at 94°C. Illumina RAP plate adaptors were used during the course of library construction. The pre-enrichment library was eluted in a final volume of 20 μ L, and 10 μ L was used in the enrichment PCR, which was a 12 cycle amplification. Post-enrichment libraries were eluted in 25 μ L of 10mM Tris, pH 8.5. The adaptor-enriched libraries were analyzed for uniformity of size using the Agilent 2200 TapeStation. The results shown in Figure 3 illustrate very consistent size distribution across all 96 samples. High Quality, Consistent Sequencing Reads After generating the 96 libraries using the TruSeq Stranded mRNA Kit, 24 of them were sequenced on two lanes of an Illumina HiSeq 2500 instrument using a TruSeq Rapid SBS 200-cycle kit. The data was then analyzed by scientists at Illumina and the Center for Genomics and Bioinformatics. The HiSeq 2500 run was successful, generating 572,468,106 reads following trimming. The base read counts (total number of reads assigned to a sample), base counts (total number of bases assigned to a sample), and the Q30 base counts (high quality base assignments) were then determined. As shown in Figure 4, the 24 sequenced libraries constructed using the Biomek FX P provided highly consistent base read counts. Figure 5 illustrates that the base counts were also highly reproducible for the stranded mRNA libraries, as the majority of the reads consist of bases with Q scores higher than 30. Figure 6 outlines the total sequence yield for each library.

Figure 3. Agilent 2200 results for 96 stranded mRNA TruSeq libraries created on the Biomek FX P

Figure 4. Pass Filter Reads for Read 1 and 2 of the Ilumina HiSeq 2500 Run.

B2013-14332 IB-18319A