Ion library quantitation kit

Total nucleic acids were extracted from l ml of frozen cryopreserved plasma using an automated NucliSens EasyMag nucleic acid extraction machine (bioMérieux, Durham, NC) followed by removal of DNA from the nucleic extract with Qiagen AllPrep DNA/RNA mini kit and ribosomal RNA by a Low Input RiboMinus System (Life Technologies). The cDNA Library was then constructed using an Ion Torrent Total RNA-Seq Kit (Life Technologies) for whole transcriptome libraries and Barcodes 1 through 8 from an Ion Xpress 1–16 barcoding kit were used (Life Technologies) for each individual sample. cDNA libraries were quantified by qPCR using an Ion Library Quantitation Kit (Life Technologies) to determine a suitable template dilution factor for subsequent emulsion PCR and sequencing.
Four barcoded samples were combined for one sequencing reaction. Template preparation for sequencing was conducted using the OneTouch Ion™ Template Kit in the OneTouch machine (Life Technologies). Ion Torrent sequencing was conducted using the Ion Proton Sequencing Kit (Life Technologies) on an Ion Proton Machine (Life Technologies) using a P1(v2)-chip (Life Technologies). The constructed cDNA was also used for HIV viral load measurement using a quantitative-real time PCR for HIV gag RNA, with a sensitivity of 10 copies/mL [26 (link)].

The IAV HA were sequenced by NGS using the method published before^{52 (link)}. Briefly, virus RNA was extracted from nasal swabs or cultured virus using QIAamp Viral RNA Mini Kit (Qiagen, #52906). Influenza A or B multiplex RT-PCR reactions were set up using 3 µl of RNA and 3 µl of primer cocktail (Supplementary Table 5) with SuperScript III one-step RT-PCR system with Platinum Taq high-fidelity DNA polymerase (Invitrogen, #12574030). PCR products were further fragmented to 200 bp with Ion Xpress Plus fragment library kit (Life Technologies, #4471269) and then ligated to Ion Xpress barcode adapters 1–96 (Life Technologies, #4474517), then samples with different barcodes were pooled. AMPure XP reagent (Agencourt, #A63881) was used to clean up the library pool and quantified with the Ion library quantitation kit (Life Technologies, #4468802), 10 pM of final library concentration was used to prepare template-positive Ion Sphere particles on the Ion OneTouch 2 instrument (Life Technologies), Ion 316 Chip version 2 (Life Technologies, #4488149) was used for sequencing on the Ion Torrent PGM. FluLINE pipeline was used for data analysis to produce consensus sequence^{52 (link)}. HA phylogenetic analysis with neighbor-joining trees were done using the Geneious 9.0.4 software (Biomatters Ltd).

A Qubit Fluorimeter (Invitrogen, CA, USA) was used to quantify the purified PCR products. The amplicons were then evaluated for fragment concentration using Ion Library Quantitation Kit (Life Technologies, CA, USA). The concentration was adjusted to 26 pM. The amplicons were attached to ion sphere particles (ISPs) using the Ion PGM kit Template OT2 400 (Life Technologies, CA, USA) according to the manufacturer’s protocol. Multiplexed sequencing was conducted using the 318 chip (Life Technologies, CA, USA) on the Ion Torrent Personal Genome platform (Life Technologies, CA, USA). Sequences were sorted by sample and filtered within the PGM software to remove low-quality reads. Finally, the data for each sample were exported to an individual Fastq file.