DOI: 10.5061/dryad.k0p2ngffn
title: .bam alignment files of Illumina and ONT sequencing of pREF plasmid

description: The expression of genes encompasses their transcription into mRNA followed
 by translation into protein. In recent years, next-generation sequencing
 and mass spectrometry methods have profiled DNA, RNA and protein abundance
 in cells. However, there are currently no reference standards that are
 compatible across these genomic, transcriptomic and proteomic methods, and
 provide an integrated measure of gene expression. Here, we use synthetic
 biology principles to engineer a multi-omics control, termed pREF, that
 can act as a universal molecular standard for next-generation sequencing
 and mass spectrometry methods. The pREF sequence encodes 21 synthetic
 genes that can be in vitro transcribed into spike-in mRNA controls, and in
 vitro translated to generate matched protein controls. The synthetic genes
 provide qualitative controls that can measure sensitivity and quantitative
 accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF
 in metagenome DNA sequencing and RNA sequencing experiments and evaluate
 the quantification of proteins using mass spectrometry. Unlike previous
 spike-in controls, pREF can be independently propagated and the synthetic
 mRNA and protein controls can be sustainably prepared by recipient
 laboratories using common molecular biology techniques. Together, this
 provides the first universal synthetic standard able to integrate genomic,
 transcriptomic and proteomic methods.
description: Illumina DNA sequencing <em>pREF</em>.
 We first sequenced neat preparations of
 <em>pREF</em>. Four replicate libraries were prepared using
 the KAPA HyperPlus PCR-based<em> </em>kit (Illumina) according
 to the manufacturer’s instructions. Prepared libraries were quantified on
 a Qubit (Invitrogen) and verified on the Agilent 2100 Bioanalyzer with the
 Agilent High Sensitivity DNA Kit (Agilent Technologies). The libraries
 were then sequenced on a NovaSeq (Illumina). The sequencing was performed
 at the Kinghorn Centre for Clinical Genomics, Darlinghurst, New South
 Wales. ONT DNA sequencing
 <em>pREF</em>. <em>pREF
 </em>was linearised using restriction enzymes, and four replicate
 libraries were prepared for nanopore sequencing, with the LSK108 kit (1D
 ligation) according to the manufacturer’s instructions. The resulting
 libraries were sequenced on a PromethION instrument, at the Kinghorn
 Centre for Clinical Genomics, Darlinghurst, New South Wales. Base-calling
 was achieved using ONT Albacore Sequencing Pipeline Software (version
 1.2.6). <em>pREF</em> alignment and kmer
 analysis. The four replicate Illumina short read DNA
 libraries were aligned to reference sequences containing the
 <em>pREF</em> plasmid using BWA-MEM2, while the two Illumina
 RNA libraries (transcribed with Sp6 and T7 polymerase) were aligned using
 bowtie2 (v2.4.0). Long read DNA and RNA libraries generated by Oxford
 Nanopore sequencing were aligned to the <em>pREF</em>
 reference sequence using MiniMap2 (v2.17-r941) with the parameters
 'minimap2 -ax map-ont' optimized for Oxford Nanopore libraries.
 Alignment files were sorted and indexed using samtools (v1.9) and
 pysamstats were used to retrieve the coverage and specific error types,
 such as mismatches or insertions and deletions, for every reference
 sequence position.
description: This README file was generated on 2024-01-25 by Helen Gunter GENERAL
 INFORMATION 1\. Title of Dataset: bam alignment files of Illumina and ONT
 sequencing of pREF plasmid 2\. Author information A. Principle
 Investigator Contact Information Name: Timothy Mercer Institution:
 Australian Institute for Bioengineering and Nanotechnology, The University
 of Queensland Address: Brisbane, Qld, Australia Email:
 B. Co-investigator Name: Helen Gunter
 Institution: Australian Institute for Bioengineering and Nanotechnology,
 The University of Queensland Address: Brisbane, Qld, Australia Email:
 3\. Date of data collection: 2021 4\.
 Geographic location of data collection: Sydney, Australia 5\. Information
 about funding sources that supported the collection of the data: NHMRC
 grants APP1108254, APP1114016, APP1136067, UNSW Tuition Fee Scholarship
 and Cancer Institute NSW Early Career Fellowship 2018/ECF013. \---
 SHARING/ACCESS INFORMATION 1\. Licenses/restrictions placed on the data:
 CC0 1.0 Universal (CC0 1.0) Public Domain 2\. Links to publications that
 cite or use the data: Gunter, H. M., et al. (in editorial review) A
 universal molecular control for DNA, mRNA and protein expression. Nat.
 Commun. 3\. Links to other publicly accessible locations of the data: None
 4\. Links/relationships to ancillary data sets: Genomic and transcriptomic
 sequencing data generated from generated from pREF:
 5\. Was data
 derived from another source? No A. If yes, list source(s): NA 6\.
 Recommended citation for this dataset: Gunter, H. M., et al. (in editorial
 review) A universal molecular control for DNA, mRNA and protein
 expression. Nat. Commun. DATA & FILE OVERVIEW 1\. File List: A)
 ONT_DNApREF_sorted.bam.bai B) ONT_DNApREF_sorted.bam C)
 Illumina_DNApREF_1mil.sorted.bam.bai D) Illumina_DNApREF_1mil.sorted.bam
 2\. Relationship between files, if important: A)
 ONT_DNApREF_sorted.bam.bai is an index file for ONT_DNApREF_sorted.bam B)
 llumina_DNApREF_1mil.sorted.bam.bai is an index file for
 Illumina_DNApREF_1mil.sorted.bam C) ONT_DNApREF_sorted.bam and
 Illumina_DNApREF_1mil.sorted.bam include sequences of the same pREF
 plasmid, generated using different sequencing chemistries 3\. Additional
 related data collected that was not included in the current data package:
 Genomic and transcriptomic sequencing data generated from generated from
 pREF: 4\. Are
 there multiple versions of the dataset? No A. If yes, name of file(s) that
 was updated: NA i. Why was the file updated? NA ii. When was the file
 updated? NA
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: ONT_DNApREF_sorted.bam 1\. Sample type
 sequenced: pREF plasmid 2\. Library preparation method: Ligation
 Sequencing 3\. Sequencing platform used: Oxford Nanopore 4\. Data
 filtering: trimmed, passed reads 5\. Aligned to: pREF plasmid sequence
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: Illumina_DNApREF_1mil.sorted.bam 1\. Sample
 type sequenced: pREF plasmid 2\. Library preparation method: KAPA
 HyperPlus PCR-based kit 3\. Sequencing platform used: Illumina 4\. Data
 filtering: trimmed, passed reads 5\. Aligned to: pREF plasmid sequence
 \#########################################################################