Sequencing library preparation

Sequencing libraries were prepared from extracted genomic DNA with the commercially available LoopSeq kits from Loop Genomics (protocols available at loopgenomics.com). Synthetic long reads (SLRs) were constructed from the short-read sequencing reads using the standard Loop Genomics informatics pipeline. The process involves attaching two DNA tags: one Unique Molecular Identifier (UMI) to each unique “parent” molecule and one sample-specific tag (i.e., a sample index) equally to all molecules in the same sample. Barcoded molecules are amplified, multiplexed, and each UMI is distributed intramolecularly to a random position within each parent molecule. Molecules are then fragmented into smaller units at the position of each UMI, creating a library of UMI-tagged fragments with an average length of 400 bp compatible with an Illumina sequencing platform run in PE150 mode.

For each LoopSeq Microbiome 16S kit, up to 24 samples were processed in multiplex and ~12,000 1.5 kb molecules were sequenced per sample (~300 k molecules from a complete kit run). 100-150M PE150 reads (50-75M clusters passing filter) were used for each sequencing run, yielding ~20 gigabases (Gb) of data. The complete sample preparation and sequencing protocol can be found in this link.

For each LoopSeq Mycobiome 18S-ITS kit, up to 24 samples were processed in multiplex and ~12,500~2.3 kb molecules were sequenced per sample (~300 k molecules from a complete kit run). 175-250M PE150 reads (87.5-125M clusters passing filter) were used for each sequencing run, yielding ~35 gigabases (Gb) of data. The complete sample preparation and sequencing protocol with sequencing instructions can be found in this link.

For each LoopSeq Bacterial Genome kit, up to 8 samples were processed in multiplex and ~40,000~5 kb molecules were sequenced per sample (~320 k molecules per library). 320M PE150 reads (160M clusters passing filter) were used for each sequencing run, yielding ~50 gigabases (Gb) of data. The complete sample preparation and sequencing protocol with sequencing instructions can be found in this link.