NEXTFLEX^® Combo-Seq^™ mRNA/miRNA Kit

Facilitating Both High- and Low-throughput Needs for Illumina^® Multiplexing

Eight UDI barcoded primers are included in the small reaction size NEXTFLEX^® Combo-Seq^™ kit and forty-eight UDI barcoded primers are included in the large reaction size kit. UDIs read as single index reads on certain instruments, including the Illumina^® MiniSeq^®, NextSeq^®, HiSeq^® 3000 or HiSeq^® 4000 platforms. If interested in reading both indices when using these sequencers, contact [email protected] for possible solutions.

Figure 1. NEXTFLEX^® Combo-Seq mRNA/miRNA kit discovers ~60% more transcripts compared to Competitor N’s mRNA-seq workflow. Libraries were generated from 20 ng of Biochain Human Universal RNA (R4234565) using the NEXTFLEX^® Combo-Seq mRNA/miRNA Kit and Competitor N’s directional mRNA-seq workflow. Sequencing libraries were downsampled to 10M reads and processed using the exceRpt pipeline (https://hub.docker.com/r/rkitchen/excerpt). Pipeline output files were used to enumerate ”transcripts discovered.” Transcripts were only marked “discovered” if >= 3 reads mapped to a given transcript and if each read aligned in the appropriate direction (sense for the NEXTFLEX^® kit and antisense for Competitor N’s kit). For “long RNA,” transcripts were counted using the “geneLevel” read counts files. In the figure, pink indicates transcripts which were detected by both workflows, whereas purple and red refer to NEXTFLEX^® and Competitor N, respectively.

Figure 2. The NEXTFLEX^® Combo-Seq^™ mRNA/miRNA kit gives higher confidence in strand calls compared to Competitor N’s directional mRNA-seq workflow. Reads were mapped to synthetic ERCC’s to determine orientation. NEXTFLEX^® chemistry has >99.9% directionality whereas Competitor N is only ~99% directional (data not shown), which allows for higher directionality/ strandedness and lower rate of false-positives regarding directionality.

Figure 3. The NEXTFLEX^® Combo-Seq^™ mRNA/miRNA kit gives more accurate representation of miRNAs than Competitor N’s small RNA-seq kit. In order to assess bias, libraries were generated from a pool of 963 equimolarly pooled miRNAs using the NEXTFLEX^® Combo- Seq^™ mRNA/miRNA kit and a commercially available small RNA-seq kit from Competitor N. Individual miRNAs were binned into categories based on their representation in the sequencing library in terms of fold-change from expected. With the NEXTFLEX^® Combo-Seq^™ mRNA/miRNA kit, only 5.8% of miRNAs deviate more than 10-fold from the expected value, compared with 30.1% of miRNAs with Competitor N.