spreading-of-signals

Index switching causes “spreading-of-signal” among multiplexed samples in Illumina HiSeq 4000 DNA sequencing.  bioRxiv  doi: https://doi.org/10.1101/125724

Exclusion Amplification chemistryという新しいシステムが使われている Illumina HiSeq 3000/4000/X Ten によるマルチプレックスシーケンスで、リードの５〜１０％が誤ったバーコードに割り振られる可能性のあることが判明したんだそうな。ウチもついこないだ外注していたRNA seqのデータを受け取ったばかりなんだけど、やっぱりHiSeq 4000が使われていた。。。このエラーによる影響の大きさは、NGSのデータを用いて実際に何をどのように解析しようとしているのかということにもよるのだろうけど、もしこれが本当なら世界中で結構大きな騒ぎになるんじゃないのかね。

「..... We show that this promising chemistry is problematic, however, when multiplexing samples. We discovered that up to 5-10% of sequencing reads (or signals) are incorrectly assigned from a given sample to other samples in a multiplexed pool. We provide evidence that this “spreading-of-signals” arises from low levels of free index primers present in the pool. These index primers can prime pooled library fragments at random via complementary 3′ ends, and get extended by DNA polymerase, creating a new library molecule with a new index before binding to the patterned flow cell to generate a cluster for sequencing. This causes the resulting read from that cluster to be assigned to a different sample, causing the spread of signals within multiplexed samples.」

「..... Therefore, all data generated in this way must now be carefully re-examined to ensure that “spreading-of-signals” has not compromised data analysis and conclusions. Re-sequencing samples using an older technology that uses conventional bridge amplification for cluster generation, or improved library cleanup strategies to remove free index primers, can minimize or eliminate this signal spreading artifact.」