The impact of read length on quantification of differentially expressed genes and splice junction detection

The initial next-generation sequencing technologies produced reads of 25 or 36 bp, and only from a single-end of the library sequence. Currently, it is possible to reliably produce 300 bp paired-end sequences for RNA expression analysis. While read lengths have consistently increased, people have assumed that longer reads are more informative and that paired-end reads produce better results than single-end reads. We used paired-end 101 bp reads and trimmed them to simulate different read lengths, and also separated the pairs to produce single-end reads. For each read length and paired status, we evaluated differential expression levels between two standard samples and compared the results to those obtained by qPCR.

We found that, with the exception of 25 bp reads, there is little difference for the detection of differential expression regardless of the read length. Once single-end reads are at a length of 50 bp, the results do not change substantially for any level up to, and including, 100 bp paired-end. However, splice junction detection significantly improves as the read length increases with 100 bp paired-end showing the best performance. We performed the same analysis on two ENCODE samples and found consistent results confirming that our conclusions have broad application.

A researcher could save substantial resources by using 50 bp single-end reads for differential expression analysis instead of using longer reads. However, splicing detection is unquestionably improved by paired-end and longer reads. Therefore, an appropriate read length should be used based on the final goal of the study.