VZV genotyping and a search for co-infection was performed on a CSF sample from case 13, using mNGS [7 (link),8 (link)]. Total nucleic acid was extracted from 90 μL of CSF using the Zymo Quick-DNA/RNA MagBead (Zymo Cat. No. R2130) via the Agilent Bravo liquid handling robot (Santa Clara, CA, USA). The nucleic acid was then divided, with half undergoing DNAse treatment to isolate RNA for RNA sequencing (RNA-seq) and the remainder being used for DNA sequencing (DNA-Seq).
RNA-Seq libraries were prepared using the New England Biolabs’ NEBNext Ultra II RNA library preparation kit (NEB Cat No. E7770, Ipswich, MA, USA), DNA libraries were prepared using the New England Biolabs’ NEBNext® Ultra™ II DNA Library preparation kit (NEB Cat No. E7645), both using the Echo Labcyte 525 and Agilent Bravo robots with a previously described protocol. Host ribosomal RNA depletion was performed using the Qiagen QIAseq FastSelect RNA removal kit (Qiagen Cat No. 333180, Hilden, Germany) at 1:100 dilution. Pooled libraries were size selected using Ampure beads. Final libraries were then sequenced on an Illumina Novaseq 6000 (San Diego, CA, USA) using 146 base pair paired-end sequencing. The mNGS workflow events are also illustrated inFigure 1 .
RNA-Seq libraries were prepared using the New England Biolabs’ NEBNext Ultra II RNA library preparation kit (NEB Cat No. E7770, Ipswich, MA, USA), DNA libraries were prepared using the New England Biolabs’ NEBNext® Ultra™ II DNA Library preparation kit (NEB Cat No. E7645), both using the Echo Labcyte 525 and Agilent Bravo robots with a previously described protocol. Host ribosomal RNA depletion was performed using the Qiagen QIAseq FastSelect RNA removal kit (Qiagen Cat No. 333180, Hilden, Germany) at 1:100 dilution. Pooled libraries were size selected using Ampure beads. Final libraries were then sequenced on an Illumina Novaseq 6000 (San Diego, CA, USA) using 146 base pair paired-end sequencing. The mNGS workflow events are also illustrated in
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