Consistent ultra-long DNA sequencing with automated slow pipetting

Trent M Prall,David A Baker,Hailey E Bussan,Emma K Neumann,Cecilia G Shortreed,Roger W Wiseman,David H O'Connor,Julie A Karl

doi:10.1186/s12864-021-07500-w

Trent M Prall, David A Baker + Show 6 more

Open Access

https://doi.org/10.1186/s12864-021-07500-w

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Journal: BMC Genomics	Publication Date: Mar 12, 2021
Citations: 10	License type: open-access

Affiliation: University of Wisconsin–Madison

Abstract

BackgroundOxford 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.ResultsWe 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.ConclusionsSNAILS 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.

Highlights

Oxford Nanopore Technologies’ instruments can sequence reads of great length
Slow nucleic acid instrument for long sequences (SNAILS) - slow nucleic acid instrument for long sequences We hypothesized that slow pipetting reagents could be automated by rotating a pipette plunger using a DC motor
An L298N motor driver supplies current to the motor and is controlled through 3.3-V signals generated by a raspberry pi 3B+ microcontroller

Summary

Introduction

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. Long reads improve genomic assemblies by spanning repetitive regions and structural variation within the genome These regions have historically complicated alignments created solely using shorter, secondgeneration sequencing reads [2]. Illumina reads aligned to GRCh38 better than other long read sequencing technologies [4] These gaps included coding exons for 76 genes with known diseaseassociated mutations cataloged in the Human Gene Mutation Database [5]. The first telomere-totelomere assemblies of the human X and Y chromosomes were created using ONT sequencing [7, 8] In these assemblies, ultra-long reads in excess of one megabase pair (Mb) were used to bridge the previous assemblies’ centromeric gaps. ONT generated long reads have been utilized to establish more contiguous assemblies for important model organisms [9,10,11,12]

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