Technology sequence analysis upgrade#
We can’t make any specific sequencing performance promises beforehand.įor many samples it can be expected that Nanopore sequencing will generate on average longer reads and higher yields per dollar as compared to sequencing on the Pacbio Sequel (a Sequel upgrade is expected for early 2019 though). Test sequencing is the only way to fully asses the suitability of DNA samples. As a consequence we will perfom test sequencing experiments (also on the smaller MinION sequencer) with new types of samples. Thus it is possible that genomic DNAs of some species contain DNA base modifications that interfere with the current Nanopore protocols. It has been observed that genomic DNA samples from certain species (including some birds and Cnidaria) perform significantly worse than for example, mammalian samples. The QC data however allow us to rule out clearly problematic samples. For example single-strand nicks and chemical adducts could escape these methods. Please note that these methods can not fully assess the quality and suitability of the DNA samples since they assess the DNA as double-stranded molecules. The sample quality can be assessed in part by spectrometry and pulsed-field-gel-eletrophoresis (PFGE). Every nick or chemical DNA adduct has the potential to abort reads and chemical contaminants could destroy the pores. As with other single molecule sequencing technologies, the read lengths and the sequencing yields do depend on the nucleic acid sample quality. The sequence yields and run metrics will vary widely depending on the samples. The sequencing data can be used to investigate RNA-base modifications. The library preparation adds an adapter via annealing to the poly-A tail. direct RNA sequencing: this protocol is being implemented.In contrast to the PacBio Iso-Seq protocol, the data cannot be processed for high accuracy circular consensus information (CCS) though.
These data are used for isoform analysis and gene annotations. The cDNA amplicons are converted to Nanopore libraries via adapter ligation.
Technology sequence analysis full#
full length cDNA sequencing: The cDNAs will be prepared after priming from the poly-A tails (analogous to the Clontech SMART-seq protocol) and PCR amplified.It is expected that a combination of super-long-read and ultra-long read data will assist de novo genome assembly projects tremendously. This type of sequencing is already possible on the MinION sequencer where we have seen read lengths of up to 840 kb and read lengths N50 of up to 70 kb (MinION). However, the yields are expected to be significantly lower compared to the super-long reads. The goal is a significant proportion of reads with lengths longer than 100 kb. The sequencing adapters are added by a transposase instead of a ligases. Ultra-long-read DNA sequencing: The protocol is currently being implemented.The yields per flow cell can vary from 20 Gb to up to 90 Gb read lengths N50 values can reach up to 33 kb and longest reads of up to 270 kb length. Super-long-read DNA sequencing: For this protocol we shear the DNA to fragments of about 50 kb lengths using the MegaRuptor then ligate-on the sequencing adapters.Yields per PromethION flowcell can range from 20 Gb up to 130 Gb. Long-read DNA sequencing: of sheared DNA fragments from 5 kb to 20 kb.We offer different types of PromethION sequencing: We have achieved the best run metrics mentioned below with DNA isolated from human/mammalian cell cultures and mammalian blood samples. Average yields will be inversely correlated to the library molecule lengths (insert sizes). The average sequence data yields can vary widely and will mostly depend on the sample properties (please see below). We now offer HMW-DNA isolation as a service (currently for animal samples) since high quality single-molecule sequencing data depend on highest quality DNA samples. Depending on the samples and the library types, the PromethION can generate up to 100 Gb of sequence data per flow cell, and can run up to 24 flow cells simultaneously. To our knowledge we are the first academic core lab to offer such services. We offer Nanopore sequencing on the highest throughput Nanopore sequencer, the PromethION. To learn more about the technology, please see the HOW-IT-WORKS page and video below. The Nanopore sequencing data greatly enable de novo genome assemblies and structural genomic variant and transcriptome studies. For high molecular weight DNA (HMW-DNA) samples, read lengths of several hundred kb can be reached with ultra-long-read protocols. Nanopore sequencing with Oxford Nanopore Technologies (ONT) systems enables high-throughput long-read sequencing of both DNA and RNA samples.