Genomics Application Notes
High Coverage Uniformity On-target sequencing must also be efficient, with uniform coverage across the transcriptome, few duplicates and polyA only fragments, and a high percentage of correct strand reads. Uniform coverage is assessed by analyzing the coverage for 1000 genes, 5' to 3', and averaging the base counts per position. The median value and CV for each position across the length of each of the 1000 genes is calculated, and then all 1000 genes are plotted together and the median coefficient of variation (median CV in Table 4) is reported. The lower the median CV, the more even the coverage, and the less probability of 5' or 3' bias. Table 4 illustrates the coverage uniformity of the sequencing results for the human libraries, as well as low percentages of poly A-only fragments and duplicates. Duplicates in mRNA sequencing must be interpreted carefully, as one gene may have multiple transcripts or duplicates may be created during PCR at the adaptor enrichment step, and these can be resolved by paired end sequencing. The correct strand percentages are very high for the stranded mRNA libraries.
Table 4. Additional Sequencing Results
POLYA (% OF PF READS)
CORRECT STRAND (% OF ALIGNED READS) PF READ COUNT
PF BASES > Q30 (% OF PF BASES)
DUPLICATES (%)
MEDIAN CV COVERAGE
PF BASES (GB)
UHR LIBRARY READ
UHR2-1 ssRNA TruSeq Library UHR2-2 ssRNA TruSeq Library UHR2-3 ssRNA TruSeq Library UHR2-4 ssRNA TruSeq Library UHR2-5 ssRNA TruSeq Library UHR2-7 ssRNA TruSeq Library UHR2-8 ssRNA TruSeq Library UHR2-9 ssRNA TruSeq Library UHR2-1 ssRNA TruSeq Library UHR2-2 ssRNA TruSeq Library UHR2-3 ssRNA TruSeq Library UHR2-4 ssRNA TruSeq Library UHR2-5 ssRNA TruSeq Library UHR2-7 ssRNA TruSeq Library UHR2-8 ssRNA TruSeq Library UHR2-9 ssRNA TruSeq Library
1
0.15%
1.53%
0.5242
99.28%
9,108,330 0.6831
93.06%
1
0.14%
1.35%
0.5105
99.43%
11,269,877 0.8452 93.60%
1
0.16%
1.46%
0.5347
99.39%
4,570,360 0.3428 93.32%
1
0.15%
1.45%
0.5195
99.36%
9,870,498 0.7403 92.95%
1
0.14%
1.62%
0.5217
99.38%
11,788,148 0.8841
92.72%
1
0.14%
1.79%
0.5364
99.52%
14,299,293 1.0724 92.51%
1
0.14%
1.56%
0.5174
99.45%
14,186,244 1.064
92.89%
1
0.14%
1.53%
0.5197
99.39%
12,493,267 0.937
92.80%
2
0.27%
1.53%
0.5242
99.28%
9,108,330 0.6831
88.38%
2
0.27%
1.35%
0.5105
99.43%
11,269,877 0.8452 89.71%
2
0.27%
1.46%
0.5347
99.39%
4,570,360 0.3428 89.44%
2
0.28%
1.45%
0.5195
99.36%
9,870,498 0.7403 88.75%
2
0.29%
1.62%
0.5217
99.38%
11,788,148 0.8841
88.09%
2
0.28%
1.79%
0.5364
99.52%
14,299,293 1.0724 87.62%
2
0.25%
1.56%
0.5174
99.45%
14,186,244 1.064
87.65%
2
0.30%
1.53%
0.5197
99.39%
12,493,267 0.937
88.30%
B2013-14332 IB-18319A
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