Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
EVALUATION AND OPTIMIZATION OF THE
ILLUMINA NEXTSEQ 500 SYSTEM FOR NEXT
GENERATION SEQUENCING (NGS)-BASED
SURVEILLANCE OF INFLUENZA
Wednesday, 1st June 20:00 La Fonda Mezzanine (2nd Floor) Poster (PS‐2b.02)
Background‐
Shoshona Le, Thomas Stark, Samuel Shepard, Elizabeth Neuhaus,
David E Wentworth, John Barnes 1
Centers for Disease Control and Prevention
The CDC Influenza Division (ID) currently utilizes Next Generation Sequencing (NGS) techniques
to conduct genetic surveillance of circulating influenza virus strains. As a World Health Organization
(WHO) Collaborating Center, ID receives between 8,000 and 10,000 specimen submissions per year
for surveillance of influenza. ID has implemented a high throughput pipeline to meet this demand
that is based on processing 96 influenza samples/controls per run across two Illumina MiSeq
instruments, to generate all of the sequencing data used for influenza genetic characterization. For
the Northern Hemisphere influenza 2015‐2016 season (October‐April), ID generated complete
influenza genomes of over 3,500 viruses using this NGS pipeline, totaling more than 30,000 genome
segments available for influenza analysis. During the peak of the influenza season, the high volume of
influenza samples resulted in 12 MiSeq runs during a single month, with a large number of
specimens still awaiting processing. The Illumina NextSeq 500 was procured in an effort to mitigate
this issue, as it has 10‐15 times more data output of the current MiSeq in roughly the same run
time. Because the NextSeq instrument differs significantly from the MiSeq instrument in the method
of measuring fluorescence signals from the flowcell, assessments were conducted to determine whether
the NextSeq would have any adverse effects on the influenza genetic data upon integration into the
ID pipeline.
Methods‐
To compare the data quality between the MiSeq and the NextSeq 500 instruments, libraries that
had been prepared and previously run on the Miseq were re‐sequenced on the NextSeq, utilizing a
loading workflow that was modified according to Illumina specifications. Pools of 96 samples that
had been barcoded using an Illumina single index kit were run, then expanded to quad‐pools of 384
samples that had been barcoded utilizing Illumina Nextera A, B, C, and D 96‐index kits. Quality
of the data and quantity of the reads were compared between the instruments, as well as consensus
sequences, subpopulations, and coverage of the influenza genomes sequenced. Further comparisons
between NextSeq v1 and NextSeq v2 chemistry were also examined.
Conclusions‐
The NextSeq 500 System offers a cost efficient NGS‐based strategy for increasing the capacity of
influenza NGS pipelines, and likely other viral pipelines, without sacrificing data quality or quantity.
Its optimizable workflow, capability to provide a 10‐fold increase in data generation, and capacity
to quadruple sample number per sequencing run, yet improve coverage enables high throughput
sequencing to meet the demand for global surveillance of the influenza A and B viruses at less than
half the cost of multiple MiSeq runs.
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