Genomics Application Notes
Seven Illumina TruSeq DNA PCR-Free libraries were sequenced on an Illumina MiSeq ® 2x 1 0 1 cycle paired end run, which generated over 9 million pass filter reads from the Illumina TruSeq DNA PCR-Free libraries. The reads were then mapped to the human reference genome (hg 1 9) using Bowtie 2 mapping software implemented on the public instance of Galaxy (usegalaxy.org) using default parameters. Mapping metrics are presented in Figure 7. Following mapping, insert size distributions were calculated for each of the 7 libraries using the Picard Insertion Size Metrics tool, also installed on the public instance of Galaxy. Median size insert values for the 7 libraries were 386±6 bp. Standard deviations for the 7 libraries were 86±2 bp. A representative size insert distribution is presented in Figure 8.
Fig. 4. Human Reference gDNA following shearing on the Covaris S220.
Following library construction, the quality and size distribution of the libraries was analyzed using the Agilent DNA High Sensitivity kit (5067-4626) and Agilent 2 1 00 Bioanalyzer. Representative Bioanalyzer 2 1 00 results are shown in Figure 5. The library concentrations were measured using KAPA SYBR Fast Universal 2X qPCR Master Mix library quantification kits (P/N KK4824, Kapa Biosystems), and are summarized in Figure 6.
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IlluminaTruSeq DNA PCR-Free Library
Fig. 7. Bowtie 2 mapping results for Illumina TruSeq DNA PCR-Free libraries.
Fig. 5. Illumina TruSeq DNA PCR-Free libraries constructed with the Biomek FX. P
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Fig. 6. Sequence-ready library yields as determined by qPCR. Mean library yield was 9.3 nM.
Fig. 8. Size insert distribution calculated using Picard Insertion Size Metrics tool for Illumina TruSeq DNA PCR-Free library 1 . Insert Size
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