Consistent ultra-long DNA sequencing with automated slow pipetting

Abstract

Background Oxford Nanopore Technologies’ instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing. Results We have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries’ average read length over manual slow pipetting. Conclusions SNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.

Long-read sequencing to resolve the parent of origin of a de novo pathogenicUBE3Avariant

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“…The application of suction force during pipetting can cause shearing of nucleic acids. To obtain longer reads, it is recommended to use tapping for mixing and employ cut-tips for pipetting, while also pipetting at a slow pace (16). Most of the DNA isolation kits are column or particle-based.…”

Epigenetic tumor heterogeneity in the era of single-cell profiling with nanopore sequencing

“…To sequence unambiguously spanning repetitive elements of the genome, long reads are required for increasing a significant length [ 187 , 188 ]. The method of pipetting reagents as slowly as possible to minimize shearing force and preserve long DNA templates during library preparation was developed and called SNAILS (a slow nucleic acid instrument for long sequences) [ 187 ]. SNAILS implements automating the slow pipetting of library preparation reagents to increase the consistency and throughput of long-read nanopore sequencing [ 187 , 189 ].…”

“…The method of pipetting reagents as slowly as possible to minimize shearing force and preserve long DNA templates during library preparation was developed and called SNAILS (a slow nucleic acid instrument for long sequences) [ 187 ]. SNAILS implements automating the slow pipetting of library preparation reagents to increase the consistency and throughput of long-read nanopore sequencing [ 187 , 189 ]. Focusing on DNA extraction and enzymatic reactions to further increase the read length, it is possible to transform from 50 to 70 kb of mechanical shearing to 90 to 100 kb reads of transposase-mediated fragmentation [ 190 ].…”