Journal: bioRxiv

Article Title: Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

doi: 10.1101/645903

Figure Lengend Snippet: Outlier consensus sequences are shown for the Bacillus subtilis reference. The data is divided by intragenomic operons, and dots signify errors annotated as mismatch, deletion or insert by color. Manual annotations can be seen to the left of the sequences. Chimeras, contamination and artefacts could not be reliably detected by software alone. Therefore, the outliers were flagged depending on error rate and with uchime2_ref chimera detection, and were manually inspected and curated: sequences with errors concentrated in one part of the sequence were flagged as chimeras, sequences with many errors and with a better hit in the SILVA database compared to the ZymoBIOMICS reference were flagged as contamination and sequences with long homopolymer inserts in the PacBio data were flagged as CCS artefacts.

Article Snippet: The ZymoBIOMICS Microbial Community DNA Standard (D6306, lot no. ZRC190811) was obtained from Zymo Research (Irvine, California).

Techniques: Software, Sequencing

Journal: bioRxiv

Article Title: Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

doi: 10.1101/645903

Figure Lengend Snippet: Each line plot represents the read size distribution from each mock community species estimated from the Nanopore metagenome data. A) is data generated from a ZymoBIOMICS Microbial Community Standard (even) [D6300, batch ZRC190633] by the Loman lab using the Nanopore GridION and a R9.4.1 flowcell. B) is in-house data generated from a ZymoBIOMICS Microbial Community DNA Standard (even) [product D6306, batch ZRC190811] using Nanopore MinION and a R10 flowcell. Some species have significantly more high molecular weight DNA over 5000 bp compared to some of the other species, which impacts the effective template availability in PCR for long amplicons. For the Loman lab data, the distinct gram+/- dependent trends fragment length is anticipated from their two step extraction protocol used on the mock community. The in-house generated data is generated from DNA standard prepared by the vendor, and here the taxa trend is not as pronounced, but the effect on the effective relative abundance is still dramatic . We can only speculate about the cause, but different extraction methods could be the reason. If all samples had been extracted with the same method, the result should be more similar fragment distributions as indicated by the Loman group data. The Nanopore library preparation and sequencing likely had an impact on the observed read fragment distributions, but the Loman group data indicates that extraction method probably is the most important factor.

Article Snippet: The ZymoBIOMICS Microbial Community DNA Standard (D6306, lot no. ZRC190811) was obtained from Zymo Research (Irvine, California).

Techniques: Generated, Molecular Weight, Sequencing

Journal: bioRxiv

Article Title: Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

doi: 10.1101/645903

Figure Lengend Snippet: Read coverage profiles of the ZymoBIOMICS Microbial Community DNA Standard based on shotgun Nanopore sequencing data. Each grey point is the average coverage value of a 10 kbp region within the genome. Colored points represents the position of the individual intragenomic rRNA operons. A) is data generated from a ZymoBIOMICS Microbial Community Standard (even) [product D6300, batch ZRC190633] by the Loman lab using the Nanopore GridION and a R9.4.1 flowcell. B) is our data generated from a ZymoBIOMICS Microbial Community DNA Standard (even) [product D6306, batch ZRC190811] using Nanopore MinION and a R10 flowcell.

Article Snippet: The ZymoBIOMICS Microbial Community DNA Standard (D6306, lot no. ZRC190811) was obtained from Zymo Research (Irvine, California).

Techniques: Nanopore Sequencing, Generated