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