A reference genome, mitochondrial genome and associated transcriptomes for the critically endangered swift parrot (<i>Lathamus discolor</i>)

Luke W. Silver; Dejan Stojanovic; Katherine A. Farquharson; Lauren Alexander; Emma Peel; Katherine Belov; Carolyn J. Hogg

doi:10.12688/f1000research.144352.2

Home Browse A reference genome, mitochondrial genome and associated transcriptomes...

ALL Metrics

Views

Downloads

Get PDF

Get XML

Export

▬

✚

Genome Note

Revised

A reference genome, mitochondrial genome and associated transcriptomes for the critically endangered swift parrot (Lathamus discolor)

[version 2; peer review: 2 approved, 1 approved with reservations]

Luke W. Silver¹, Dejan Stojanovic², Katherine A. Farquharson^1,3, [...] Lauren Alexander^1,3, Emma Peel^1,3, Katherine Belov^1,3, Carolyn J. Hogg ^1,3

Luke W. Silver¹, Dejan Stojanovic², [...] Katherine A. Farquharson^1,3, Lauren Alexander^1,3, Emma Peel^1,3, Katherine Belov^1,3, Carolyn J. Hogg ^1,3

PUBLISHED 27 Aug 2024

Author details Author details

¹ School of Life and Environmental Sciences, The University of Sydney, Sydney, New South Wales, 2006, Australia
² Fenner School of Environment and Society, Australian National University, Acton, Australian Capital Territory, 2601, Australia
³ Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science,, The University of Sydney, Sydney, New South Wales, 2006, Australia

Luke W. Silver
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation

Dejan Stojanovic
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Resources, Writing – Review & Editing

Katherine A. Farquharson
Roles: Methodology, Supervision, Writing – Review & Editing

Lauren Alexander
Roles: Data Curation, Methodology, Writing – Review & Editing

Emma Peel
Roles: Methodology, Writing – Review & Editing

Katherine Belov
Roles: Conceptualization, Funding Acquisition, Supervision, Writing – Review & Editing

Carolyn J. Hogg
Roles: Conceptualization, Funding Acquisition, Project Administration, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Genomics and Genetics gateway.

Abstract

Abstract*

The swift parrot (Lathamus discolor) is a Critically Endangered migratory parrot that breeds in Tasmania and winters on the Australian mainland. Here we provide a reference genome assembly for the swift parrot. We sequence PacBio HiFi reads to create a high-quality reference assembly and identify a complete mitochondrial sequence. We also generate a reference transcriptome from five organs to inform genome annotation. The genome was 1.24 Gb in length and consisted of 847 contigs with a contig N50 of 18.97 Gb and L50 of 20 contigs. This study provides an annotated reference assembly and transcriptomic resources for the swift parrot to assist in future conservation genomic research.

Keywords

Genome assembly, reference genome, transcriptome, Aves, mitogenome

Corresponding author: Carolyn J. Hogg

Competing interests: No competing interests were disclosed.

Grant information: Sequencing received in-kind support from the NCRIS funded BioPlatforms Threatened Species Initiative are supported by the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012), Australian BioCommons which is enabled by NCRIS via Bioplatforms Australia and the University of Sydney.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Silver LW et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Silver LW, Stojanovic D, Farquharson KA et al. A reference genome, mitochondrial genome and associated transcriptomes for the critically endangered swift parrot (Lathamus discolor) [version 2; peer review: 2 approved, 1 approved with reservations]. F1000Research 2024, 13:251 (https://doi.org/10.12688/f1000research.144352.2) First published: 04 Apr 2024, 13:251 (https://doi.org/10.12688/f1000research.144352.1) Latest published: 27 Aug 2024, 13:251 (https://doi.org/10.12688/f1000research.144352.2)

Revised Amendments from Version 1

We have addressed the reviewers comments around the mitogenome assembly and addressed the other minor comments.

See the authors' detailed response to the review by Charles Feigin
See the authors' detailed response to the review by Phred Benham

Introduction

The swift parrot (Lathamus discolor) is a migratory parrot that breeds on the eastern seaboard of the island of Tasmania, Australia and winters on southeastern mainland Australia (Kennedy & Tzaros, 2005; MacNally & Horrocks, 2000; Saunders & Heinsohn, 2008). The swift parrot is Critically Endangered (BirdLife International, 2018) due to the combined effects of logging of its important breeding habitat (Webb et al., 2019) and the impacts of an introduced predator, the sugar glider (Petaurus breviceps) (Heinsohn et al., 2015). Population viability analysis has shown that the already small population of only a few hundred swift parrots (Olah et al., 2021) is likely to rapidly decline over coming generations (Heinsohn et al., 2015; Owens et al., 2023) Although the species has already been subject to population genetic study (Olah et al., 2021; Stojanovic et al., 2018), there remain outstanding questions about multiple aspects of the species’ genetic ecology. For example, like other parrots with small population sizes (Morrison et al., 2020), understanding the genetic basis of immune competence is critical for managing demographic impacts of disease in swift parrots (Saunders & Tzaros, 2011). To facilitate detailed genomic research on this species, we sequenced DNA with PacBio long reads to generate a draft reference assembly and sequenced RNA from five tissues to provide transcriptomic resources to assist in genome annotation for the swift parrot.

Methods

Sample collection and DNA/RNA extraction

A single captive bred female swift parrot died as a result of liver infection. Tissue samples were dissected and flash frozen at -80°C or preserved in RNAlater before being frozen at -80°C. High molecular weight (HMW) DNA was then extracted from heart and kidney tissue using the Nanobind Tissue Big DNA Kit v1.0 (Circulomics: SKU 102-302-100) using the standard protocol. A Qubit fluorometer was used to assess the concentration of DNA with the Qubit dsDNA BR assay kit (Thermo Fisher Scientific). Total RNA was extracted from gonad, spleen, liver, heart and kidney using the RNeasy Plus Mini Kit (Qiagen: 74134) with RNAse-free DNAse I set (Qiagen: EN0521) using the standard protocol. RNA quality was determined using the NanoDrop (Thermo Fisher Scientific) and RNA integrity (RIN) score determined using the Bioanalyzer RNA nano 6000 kit (Agilent 2100: 5067-1511).

Library construction and sequencing

HMW DNA was sent for Pacific Biosciences High Fidelity (PacBio HiFi) library preparation with the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences: 101-853-100) and sequencing on one single molecule real-time (SMRT) cell of the PacBio Sequel II at the Australian Genome Research Facility (St Lucia, Australia). Total RNA from the heart, gonad, kidney, liver and spleen was sequenced as 100 bp paired-end (PE) reads using an Illumina Novaseq 6000 with Illumina Stranded mRNA library preparation at the Ramaciotti Centre for Genomics (University of New South Wales, Kensington, Australia).

Genome assembly

The genome assembly was conducted on the Galaxy Australia public server usegalaxy.org.au (Afgan et al., 2016) running the Genome assembly with ‘hifiasm’ (RRID:SCR_021069) (Cheng et al., 2022) on Galaxy Australia workflow v2.1 (Price & Farquharson, 2022). Briefly, Picard (http://broad institute.github.io/picard) (Galaxy version 2.18.2.2; RRID:SCR_006525) SamToFastq, samtools (Danecek et al., 2021; Li et al., 2009) (Galaxy version 2.0.3; RRID:SCR_002105) flagstat and fastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (Galaxy version 0.72; RRID:SCR_014583) was used to convert BAM files to FASTQ and quality check the reads for input to Hifiasm (Cheng et al., 2022). Hifiasm, with default parameters (Galaxy version 2.1), was run on Galaxy Australia to assembly the genome. Basic genome assembly statistics were calculated with the stats.sh script in BBMap (sourceforge.net/projects/bbmap/) (RRID:SCR_016965). Genome completeness was determined using Benchmarking Universal Single-Copy Orthologues (BUSCO; RRID:SCR_015008) v5.4.6 (Simao et al., 2015) with the vertebrata_odb10 (n = 3354) and aves_odb10 (n= 8338) lineage on Galaxy Australia. Repetitive elements of the genome were identified, classified and masked using a Pawsey Supercomputing Centre Nimbus cloud machine (256GB RAM, 64 vCPU, 3 TB storage) by building a database using RepeatModeler v2.0.1 (RRID:SCR_015027) (Flynn et al., 2020); repeats were then masked using RepeatMasker v4.0.9 (RRID:SCR_012954) (Smit et al., 2013-2015) with the -nolow parameter to avoid masking low complexity repeats.

Mitochondrial assembly

The mitochondrial genome was identified from the reference genome assembly using MitoHiFi v2 (Allio et al., 2020; Uliano-Silva et al., 2023). MitoHifi identified the most taxonomically closely related publicly available mitochondrial genome as the thick-billed parrot (Rhynchopsitta pachyrhyncha) (NCBI reference sequence OR209192.1). The mitochondrial reference sequence for the thick-billed parrot was then used to search for the swift parrot mitochondrial genome. The identified mitochondrial sequence was then added to the genome assembly and annotated using MITOS v 2.1.7 (Donath et al., 2019) and visualised using Proksee (Grant et al., 2023).

Transcriptome assembly

Transcriptome assembly was performed on the University of Sydney High Performance Computer, Artemis. Raw transcriptome reads were quality assessed pre and post trimming with FastQC v0.11.8 (RRID:SCR_014583). Trimmomatic v0.39 (RRID:SCR_011848) (Bolger et al., 2014) with the parameters SLIDINGWINDOW:4:5, LEADING:5, TRAILING:5 and MINLEN:25 and ILLUMINACLIP:2:30:10 with the TruSeq3-PE adapters was used to quality trim reads. The repeat masked genome was indexed and trimmed reads aligned using the -dta parameter with hisat2 v2.1.0 (RRID:SCR_015530) (Kim et al., 2019). Resulting sam files were converted to bam format and sorted using samtools v1.9 (Danecek et al., 2021). Stringtie v2.1.6 (RRID:SCR_016323) (Pertea et al., 2015) was used to generate a GTF for each transcriptome. Stringtie v2.1.6 with the -merge parameter merged transcripts into a global transcriptome retaining only transcripts with an FPKM > 0.1 and length > 30. CPC2 v2019-11-19 (Kang et al., 2017) was used to predict coding potential and only transcripts predicted to be coding were retained. Transdecoder v2.0.1 (https://github.com/TransDecoder/TransDecoder) (RRID:SCR_017647) was used to predict open reading frames in the global transcriptome with a minimum transcript length of 20. Transcriptome completeness was assessed using BUSCO v5.4.6 (Simao et al., 2015) with the vertebrata_odb10 (n = 3354) and aves_odb10 (n = 8338) lineage on Galaxy Australia.

Genome annotation

Genome annotation was performed using FGENESH++ v7.2.2 (Softberry; RRID:SCR_018928 (Solovyev et al., 2006)) using the longest open reading frame as predicted from the global transcriptome, non-mammalian settings and optimised parameters supplied with the American crow (Corvus brachyrhynchos) gene finding matrix, which is the closest related species with a gene finding matrix provided by FGENESH++. BUSCO v5.4.6 (Simao et al., 2015) in protein mode was run on Galaxy Australia to assess the completeness of the annotation with the vertebrata_odb10 (n = 3354) and aves_odb10 (n = 8338) lineage. The ‘genestats’ script (https://github.com/darencard/GenomeAnnotation) was used to obtain the average number of exons and introns and the average exon and intron length.

Results

Genome assembly

Genome assembly using Hifiasm with PacBio HiFi data from a single SMRT cell resulted in a coverage of 28.7x and a genome of 1.24 Gb in size consisting of 847 contigs with a contig N50 of 18.97 Mb and L50 of 20 contigs. The genome assembly was also highly complete with 97.0% of aves_odb10 complete BUSCOs identified (Table 1). The mitochondrial genome was 17,265 bp long and contained 38 genes, including 22 tRNAs and 14 protein coding genes, with a GC percentage of 44.88% (Figure 1).

Table 1. Genome assembly statistics of the swift parrot (Lathamus discolor) with statistics calculated with the stats.sh script as part of the BBMap software (https://sourceforge.net/projects/bbmap/) and BUSCO (Simao et al., 2015) completeness, calculated with both the vertebrata_obd10 and aves_obd10 lineages.

Metric
Assembly size (Gb)	1.24
Number of contigs	847
Contig N50 (Mb)	18.97
Contig N90 (Mb)	2.46
Contig L50	20
Contig L90	83
Longest contig (Mb)	78.39
GC content (%)	42.8
Complete vertebrata_odb10 BUSCOs	96.3% (Single copy: 94.7%, Duplicated: 1.6%)
Fragmented vertebrata_odb10 BUSCOs	1.0%
Missing vertebrata_odb10 BUSCOs	2.7%
Complete aves_odb10 BUSCOs	97.0% (Single copy: 96.1%, Duplicated: 0.9%)
Fragmented aves_odb10 BUSCOs	0.5%
Missing aves_odb10 BUSCOs	2.5%

Figure 1. Mitochondrial genome of the swift parrot (Lathamus discolor) generated with Proksee (Grant et al., 2023).

Transcriptome assembly and genome annotation

Trimming retained greater than 99.95% of raw reads which were then aligned to the repeat-masked reference genome. Individual tissue transcriptomes had variable mapping rates from 31.04% for heart tissue to 82.76% for gonad tissue (kidney: 62.26%, liver: 78.84%, spleen: 73.60%). The alignment rate for the heart tissue was low so we excluded heart transcripts from downstream analysis. The poor performance of the heart tissue is potentially due to the comparatively lower concentration of RNA in the heart tissue extraction (35.2 ng/μl) compared to the other 4 tissues (average = 1243 ng/μl [SD: 481]) and the heart tissue was not stored in RNAlater. After using stringtie -merge to generate a global transcriptome and filtering on coding potential and open reading frames with CPC2 and transdecoder, respectively, 14,045 longest open reading frame transcripts were used as mRNA evidence to guide genome annotation. The global transcriptome had 90.8% complete aves_odb10 BUSCOs (Table 2). A total of 27,867 genes were predicted from genome annotation, higher than the predicted 15,000-16,000 genes in birds (Zhang et al., 2014). The annotation contained 78.1% complete aves_odb10 BUSCOs (Table 2). Repetitive elements comprised 17.25% of the genome, mainly consisting of long interspersed elements (LINEs), comparable with other bird genomes (Zhang et al., 2014) (Table 3).

Table 2. Statistics of the global transcriptome and annotation of the swift parrot (Lathamus discolor) including BUSCO (Simao et al., 2015) completeness, calculated with both the vertebrata_obd10 and aves_obd10 lineages and average exon length.

Metrics
Global Transcriptome
Complete vertebrata_odb10 BUSCOs	94.8% (Single copy: 33.5%, Duplicated: 61.3%)
Fragmented vertebrata_odb10 BUSCOs	1.1%
Missing vertebrata_odb10 BUSCOs	4.1%
Complete aves_odb10 BUSCOs	90.8% (Single copy: 32.8%, Duplicated 58.0%)
Fragmented aves_odb10 BUSCOs	1.1%
Missing aves_odb10 BUSCOs	8.1%
Annotation
Complete vertebrata_odb10 BUSCOs	72.5% (Single copy: 70.4%, Duplicated: 2.1%)
Fragmented vertebrata_odb10 BUSCOs	10.6%
Missing vertebrata_odb10 BUSCOs	16.9%
Complete aves_odb10 BUSCOs	78.1% (Single copy: 77.2%, Duplicated: 0.9%)
Fragmented aves_odb10 BUSCOs	5.4%
Missing aves_odb10 BUSCOs	16.5%
Average number of exons per gene	7.84
Average number of introns per gene	6.84
Average exon length (bp)	2368
Average intron length (bp)	22346

Table 3. Classification of repeat elements of the swift parrot (Lathamus discolor) genome assembly as generated by the repeatmasker software (Smit et al., 2013-2015).

Repeat element	Number of elements	% of sequence
SINEs	2917	0.03
MIRs	1302	0.01
LINES	252190	8.11
LINE1	831	0.01
LINE2	528	0
L3/CR1	250524	8.08
LTR elements	26360	1.38
ERVL	10824	0.42
ERV Class I	9460	0.56
ERV Class II	4642	0.23
DNA Transposons	4580	0.04
hAT-Charlie	334	0
Unclassified	105511	7.69
Total interspersed repeats		17.25
Small RNA	2275	0.09

Ethical considerations

The sample used for genome and transcriptome sequencing was obtained from an individual who died of natural causes.

Data availability

Underlying data

The raw PacBio HiFi and transcriptome data are publicly available through the Bioplatforms Australia Threatened Species Initiative: https://data.bioplatforms.com/organization/threatened-species . The assembled genome, global transcriptome and annotation generated in this study are available on Amazon Web Services Australasian Genomes Open Data Store: https://awgg-lab.github.io/australasiangenomes/genomes.html.

Raw genome and transcriptome sequences are also available from NCBI’s Short Read Archive (SRA) accession numbers SRR26186073 to SRR26186078 (Silver et al., 2023).

And the assembled genome from NCBI’s Assembly database, BioProject: PRJNA1021263 (Silver et al., 2023).

Reporting guidelines

Figshare: Author Checklist - ARRIVE.pdf, https://doi.org/10.6084/m9.figshare.25396294.

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Acknowledgements

Thanks to Bonorong Wildlife Sanctuary and the Department of Natural Resources and Environment Tasmania for facilitating access to genetic samples. Computational resources were provided by the Australian FGENESH++ Service provided by the Australian BioCommons and the Pawsey Supercomputing Research Centre with funding from the Australian Government and the Government of Western Australia; Galaxy Australia, a service provided by the Australian Biocommons and its partners; and the University of Sydney’s High Performance Computing facility Artemis provided by the Sydney Informatics Hub. The authors wish to acknowledge the use of the services and facilities of the Ramaciotti Centre for Genomics, UNSW and of the Australian Genome Research Facility.

References

Afgan E, Baker D, van den Beek M , et al.: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 2016; 44(W1): W3–W10. PubMed Abstract | Publisher Full Text | Free Full Text
Allio R, Schomaker-Bastos A, Romiguier J, et al.: MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics. Mol. Ecol. Resour. 2020; 20(4): 892–905. PubMed Abstract | Publisher Full Text | Free Full Text
BirdLife International: Lathamus discolor. The IUCN Red List of Threatened Species 2018.2018. Publisher Full Text
Bolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract | Publisher Full Text | Free Full Text
Cheng H, Jarvis ED, Fedrigo O, et al.: Haplotype-resolved assembly of diploid genomes without parental data. Nat. Biotechnol. 2022; 40(9): 1332–1335. PubMed Abstract | Publisher Full Text | Free Full Text
Danecek P, Bonfield JK, Liddle J, et al.: Twelve years of SAMtools and BCFtools. Gigascience. 2021; 10(2). PubMed Abstract | Publisher Full Text | Free Full Text
Donath A, Jühling F, Al-Arab M, et al.: Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Res. 2019 Nov 18; 47(20): 10543–10552. PubMed Abstract | Publisher Full Text | Free Full Text
Flynn JM, Hubley R, Goubert C, et al.: RepeatModeler2 for automated genomic discovery of transposable element families. Proc. Natl. Acad. Sci. USA. 2020; 117(17): 9451–9457. PubMed Abstract | Publisher Full Text | Free Full Text
Grant JR, Enns E, Marinier E, et al.: Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023; 51(W1): W484–W492. PubMed Abstract | Publisher Full Text | Free Full Text
Heinsohn R, Webb M, Lacy R, et al.: A severe predator-induced population decline predicted for endangered, migratory swift parrots (Lathamus discolor). Biol. Conserv. 2015; 186: 75–82. Publisher Full Text
Kang YJ, Yang DC, Kong L, et al.: CPC2: a fast and accurate coding potential calculator based on sequence intrinsic features. Nucleic Acids Res. 2017; 45(W1): W12–W16. PubMed Abstract | Publisher Full Text | Free Full Text
Kennedy SJ, Tzaros CL: Foraging ecology of the swift parrot Lathamus discolor in the box-ironbark forests and woodlands of Victoria. Pac. Conserv. Biol. 2005; 11(3): 158–173. Publisher Full Text
Kim D, Paggi JM, Park C, et al.: Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019; 37(8): 907–915. PubMed Abstract | Publisher Full Text | Free Full Text
Li H, Handsaker B, Wysoker A, et al.: The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. PubMed Abstract | Publisher Full Text | Free Full Text
MacNally R, Horrocks G: Landscape-scale conservation of an endangered migrant:the swift parrot (Lathamus discolor) in its winter range. Biol. Conserv. 2000; 92(3): 335–343. Publisher Full Text
Morrison CE, Hogg CJ, Gales R, et al.: Low innate immune-gene diversity in the critically endangered orange-bellied parrot (Neophema chrysogaster). Emu Austral Ornithol. 2020; 120(1): 56–64. Publisher Full Text
Olah G, Stojanovic D, Webb MH, et al.: Comparison of three techniques for genetic estimation of effective population size in a critically endangered parrot. Anim. Conserv. 2021; 24(3): 491–498. Publisher Full Text
Owens G, Heinsohn R, Crates R, et al.: Long-term ecological data confirm and refine conservation assessment of critically endangered swift parrots. Anim. Conserv. 2023; 26(4): 450–463. Publisher Full Text
Pertea M, Pertea GM, Antonescu CM, et al.: StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015; 33(3): 290–295. PubMed Abstract | Publisher Full Text | Free Full Text
Price G, Farquharson K: PacBio HiFi genome assembly using hifiasm v2.1. WorkflowHub. 2022. Publisher Full Text
Saunders DL, Heinsohn R: Winter habitat use by the endangered, migratory swift parrot (Lathamus discolor) in New South Wales. Emu Austral Ornithol. 2008; 108(1): 81–89. Publisher Full Text
Saunders DL, Tzaros CL: National recovery plan for the swift parrot Lathamus discolor. Melbourne: Birds Australia; 2011.
Silver LW, Stojanovic D, Farquharson K, et al.: Lathamus discolor Genome sequencing and assembly.2023. Reference Source
Simao FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31(19): 3210–3212. Publisher Full Text
Smit A, Hubley R, Green P: RepeatMasker Open-4.0.2013-2015.
Solovyev V, Kosarev P, Seledsov I, et al.: Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biol. 2006; 7 Suppl 1(Suppl 1): S10.1–S1012. PubMed Abstract | Publisher Full Text | Free Full Text
Stojanovic D, Olah G, Webb M, et al.: Genetic evidence confirms severe extinction risk for critically endangered swift parrots: Implications for conservation management. Anim. Conserv. 2018; 21(4): 313–323. Publisher Full Text
Uliano-Silva M, Ferreira JGRN, Krasheninnikova K, et al.: MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio High Fidelity reads. bioRxiv. 2023. 2022.2012.2023.521667. Publisher Full Text
Webb MH, Stojanovic D, Heinsohn R: Policy failure and conservation paralysis for the critically endangered swift parrot. Pac. Conserv. Biol. 2019; 25(2): 116–123. Publisher Full Text
Zhang G, Li C, Li Q, et al.: Comparative genomics reveals insights into avian genome evolution and adaptation. Science. 2014; 346(6215): 1311–1320. PubMed Abstract | Publisher Full Text | Free Full Text

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 04 Apr 2024

Author details Author details

Luke W. Silver
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Software, Writing – Original Draft Preparation

Dejan Stojanovic
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Resources, Writing – Review & Editing

Katherine A. Farquharson
Roles: Methodology, Supervision, Writing – Review & Editing

Lauren Alexander
Roles: Data Curation, Methodology, Writing – Review & Editing

Emma Peel
Roles: Methodology, Writing – Review & Editing

Katherine Belov
Roles: Conceptualization, Funding Acquisition, Supervision, Writing – Review & Editing

Carolyn J. Hogg
Roles: Conceptualization, Funding Acquisition, Project Administration, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

Sequencing received in-kind support from the NCRIS funded BioPlatforms Threatened Species Initiative are supported by the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012), Australian BioCommons which is enabled by NCRIS via Bioplatforms Australia and the University of Sydney.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (2)

version 2

Revised

Published: 27 Aug 2024, 13:251

https://doi.org/10.12688/f1000research.144352.2

version 1

Published: 04 Apr 2024, 13:251

https://doi.org/10.12688/f1000research.144352.1

© 2024 Silver LW et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Download

Export To

metrics

	Views	Downloads
F1000Research	-	-
PubMed Central Data from PMC are received and updated monthly.	-	-

Citations

SEE MORE DETAILS

CITE

how to cite this article

Silver LW, Stojanovic D, Farquharson KA et al. A reference genome, mitochondrial genome and associated transcriptomes for the critically endangered swift parrot (Lathamus discolor) [version 2; peer review: 2 approved, 1 approved with reservations]. F1000Research 2024, 13:251 (https://doi.org/10.12688/f1000research.144352.2)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

track

receive updates on this article

Track an article to receive email alerts on any updates to this article.

Open Peer Review

Current Reviewer Status: ?

Key to Reviewer Statuses VIEW HIDE

ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 2

VERSION 2

PUBLISHED 27 Aug 2024

Revised

Views

Reviewer Report 18 Sep 2024

Joan Ferrer Obiol, University of Milan, Milan, Italy; Universitat de Barcelona (UB), Barcelona, Spain

Approved

https://doi.org/10.5256/f1000research.170611.r318594

Silver et al. present a highly-contiguous reference genome for the Critically Endangered Swift parrot. The methods used are sound but I have a few comments and suggestions:

Abstract:
Line 5: there is a mistake on the units of the N50 and should probably be changed to Mb rather than Gb.

Introduction:
Line 4: I suggest changing "its important breeding habitat" to something more informative about the type of habitat that the species occupies.
Line 8: I suggest changing genetic ecology by a more appropriate concept such as ecological genetics.
Line 11: Consider changing "we sequenced DNA with PacBio long reads to generate a draft reference assembly" to something like "we produced a draft genome assembly for the species using PacBio long reads"

Methods:

Transcriptome assembly:
I would encourage the authors to make it clear to the reader why they decided to remove the non-coding transcripts from the transcriptome and why they decided to not use them for the annotation. The important role of long non-coding RNAs in evolution is increasingly being demonstrated (e.g. Toomey et al. 2018; Mattick et al. 2023) and thus it seems strange to ignore them.

Results:

Genome assembly:
Regarding the mitogenome, I would suggest mentioning that the mitogenome was circularized. Also, I would consider annotating the two rRNA genes and the control region in Proksee.

Transcriptome assembly and genome annotation:

Given the low BUSCO completeness of the annotation, despite the relatively high completeness of the transcriptome, I would encourage the authors to use an alternative genome annotation pipeline that makes a better use of the RNASeq data such as BRAKER3.

In addition, given the high number of genes obtained in the annotation, I would consider doing some kind of filtering of the raw annotation, e.g. using gFACs (Ref 3) to remove potentially missannotated genes.

Literature cited in the review:
Mattick, J.et.al. 2023 (Ref 1)
Toomey, M. B.et.al., 2018 (Ref 2)
Caballero, M.et.al., 2019 (Ref 3)

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

References

1. Mattick JS, Amaral PP, Carninci P, Carpenter S, et al.: Long non-coding RNAs: definitions, functions, challenges and recommendations.Nat Rev Mol Cell Biol. 2023; 24 (6): 430-447 PubMed Abstract | Publisher Full Text
2. Toomey MB, Marques CI, Andrade P, Araújo PM, et al.: A non-coding region near Follistatin controls head colour polymorphism in the Gouldian finch.Proc Biol Sci. 2018; 285 (1888). PubMed Abstract | Publisher Full Text
3. Caballero M, Wegrzyn J: gFACs: Gene Filtering, Analysis, and Conversion to Unify Genome Annotations Across Alignment and Gene Prediction Frameworks.Genomics Proteomics Bioinformatics. 2019; 17 (3): 305-310 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Population genomics, phylogenomics, ecological genomics and some experience in genome assembly

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

Report a concern

Respond or Comment

Views

Reviewer Report 10 Sep 2024

Phred Benham, University of Massachusetts Amherst Department of Biology (Ringgold ID: 117236), Amherst, Massachusetts, USA

Approved

https://doi.org/10.5256/f1000research.170611.r317694

The authors have addressed my comments and those of ... Continue reading

CITE

Report a concern

Respond or Comment

Version 1

VERSION 1

PUBLISHED 04 Apr 2024

Views

Reviewer Report 13 Jul 2024

Phred Benham, University of Massachusetts Amherst Department of Biology (Ringgold ID: 117236), Amherst, Massachusetts, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.158132.r296521

This manuscript describes a de novo assembly of the critically endangered swift parrot.
Generally the sequencing and assembly methods reflect current standards and they produce a highly contiguous, contig-level assembly for this species that will be of value for various conservation genomics and other molecular ecology questions. I have only a few minor comments.

Is there a reason you did not attempt to produce a scaffolded assembly?

If there is space it would be nice to have a figure(s) showing the parrot, distribution, etc.

I agree with the other reviewer that this assembly is larger than the typical avian mito-genome and worth confirming there is not spurious sequence included.

Was a voucher specimen preserved of the parrot that died?

The last sentence of the 'mitochondrial assembly' section is not clearly written, please revise.

Resulting sam files 'were' not 'with' converted...

Why did you use crow for the gene finding matrix. Were there not other parrots or something more closely related?

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: genomics, ornithology, evolution

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

Report a concern

Author Response 27 Aug 2024

Luke Silver, School of Life and Environmental Sciences, The University of Sydney, Sydney, 2006, Australia

27 Aug 2024

Author Response

We did not attempt a scaffolded assembly as for conservation and population genetics work we have found a long read assembly to be sufficient, additionally after we produced our long ... Continue reading We did not attempt a scaffolded assembly as for conservation and population genetics work we have found a long read assembly to be sufficient, additionally after we produced our long read assembly a chromosome level scaffolded assembly was released by VGP.

In lieu of adding in an additional figure showing the distribution of the swift parrot, we have added some additional detail as to the regions where swift parrots breed and migrate to
“The swift parrot ( Lathamus discolor) is a migratory parrot that breeds on the eastern seaboard of the island of Tasmania, Australia and winters on southeastern mainland Australia”

As requested by the other reviewer I have ensured the mitochondrial information included in the manuscript and online is correct and updated to reflect this.

The individual used for the genome and transcriptome will be accessioned into a museum collection by the end of 2024, when this has been completed the BioProject information on NCBI will be updated to provide the accession number

I have rephrased this to make sense. “…genome as, the thick-billed parrot ( Rhynchopsitta pachyrhyncha) (NCBI reference sequence OR209192.1). The mitochondrial reference sequence for the thick-billed parrot was then, used…”

I have changed with to were

The crow is the most closely related species with a matrix provided by FGENESH, I have edited the methods to reflect this. “…gene finding matrix, which is the closest related species with a gene finding matrix provided by FGENESH++”
We did not attempt a scaffolded assembly as for conservation and population genetics work we have found a long read assembly to be sufficient, additionally after we produced our long read assembly a chromosome level scaffolded assembly was released by VGP.

In lieu of adding in an additional figure showing the distribution of the swift parrot, we have added some additional detail as to the regions where swift parrots breed and migrate to
“The swift parrot ( Lathamus discolor) is a migratory parrot that breeds on the eastern seaboard of the island of Tasmania, Australia and winters on southeastern mainland Australia”

As requested by the other reviewer I have ensured the mitochondrial information included in the manuscript and online is correct and updated to reflect this.

The individual used for the genome and transcriptome will be accessioned into a museum collection by the end of 2024, when this has been completed the BioProject information on NCBI will be updated to provide the accession number

I have rephrased this to make sense. “…genome as, the thick-billed parrot ( Rhynchopsitta pachyrhyncha) (NCBI reference sequence OR209192.1). The mitochondrial reference sequence for the thick-billed parrot was then, used…”

I have changed with to were

The crow is the most closely related species with a matrix provided by FGENESH, I have edited the methods to reflect this. “…gene finding matrix, which is the closest related species with a gene finding matrix provided by FGENESH++”
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 27 Aug 2024

Luke Silver, School of Life and Environmental Sciences, The University of Sydney, Sydney, 2006, Australia

27 Aug 2024

Author Response

We did not attempt a scaffolded assembly as for conservation and population genetics work we have found a long read assembly to be sufficient, additionally after we produced our long ... Continue reading We did not attempt a scaffolded assembly as for conservation and population genetics work we have found a long read assembly to be sufficient, additionally after we produced our long read assembly a chromosome level scaffolded assembly was released by VGP.

In lieu of adding in an additional figure showing the distribution of the swift parrot, we have added some additional detail as to the regions where swift parrots breed and migrate to
“The swift parrot ( Lathamus discolor) is a migratory parrot that breeds on the eastern seaboard of the island of Tasmania, Australia and winters on southeastern mainland Australia”

As requested by the other reviewer I have ensured the mitochondrial information included in the manuscript and online is correct and updated to reflect this.

The individual used for the genome and transcriptome will be accessioned into a museum collection by the end of 2024, when this has been completed the BioProject information on NCBI will be updated to provide the accession number

I have rephrased this to make sense. “…genome as, the thick-billed parrot ( Rhynchopsitta pachyrhyncha) (NCBI reference sequence OR209192.1). The mitochondrial reference sequence for the thick-billed parrot was then, used…”

I have changed with to were

The crow is the most closely related species with a matrix provided by FGENESH, I have edited the methods to reflect this. “…gene finding matrix, which is the closest related species with a gene finding matrix provided by FGENESH++”
We did not attempt a scaffolded assembly as for conservation and population genetics work we have found a long read assembly to be sufficient, additionally after we produced our long read assembly a chromosome level scaffolded assembly was released by VGP.

In lieu of adding in an additional figure showing the distribution of the swift parrot, we have added some additional detail as to the regions where swift parrots breed and migrate to
“The swift parrot ( Lathamus discolor) is a migratory parrot that breeds on the eastern seaboard of the island of Tasmania, Australia and winters on southeastern mainland Australia”

As requested by the other reviewer I have ensured the mitochondrial information included in the manuscript and online is correct and updated to reflect this.

The individual used for the genome and transcriptome will be accessioned into a museum collection by the end of 2024, when this has been completed the BioProject information on NCBI will be updated to provide the accession number

I have rephrased this to make sense. “…genome as, the thick-billed parrot ( Rhynchopsitta pachyrhyncha) (NCBI reference sequence OR209192.1). The mitochondrial reference sequence for the thick-billed parrot was then, used…”

I have changed with to were

The crow is the most closely related species with a matrix provided by FGENESH, I have edited the methods to reflect this. “…gene finding matrix, which is the closest related species with a gene finding matrix provided by FGENESH++”
Competing Interests: No competing interests were disclosed. Close
Report a concern

Views

Reviewer Report 04 Jun 2024

Charles Feigin, Department of Environment and Genetics, La Trobe University (Ringgold ID: 2080), Melbourne, Victoria, Australia

Approved with Reservations

https://doi.org/10.5256/f1000research.158132.r274647

Here, the authors present a short report detailing the production of nuclear and mitochondrial genome assemblies for the swift parrot Lathamus discolor, together with annotations supported by multi-tissue transcriptomes. The swift parrot is a Critically Endangered species and the target of conservation efforts, making these new data and resources valuable. Overall, the report is well written, and I appreciated the very detailed methods sections with parameters spelled out. I had one notable comment related to data availability and sequence length of the mitochondrial genome assembly. Further comments are fairly minor given the straightforward nature of the genome report.

Data availability and length discrepancy of the mitochondrial genome assembly - While other data in the paper has been made readily available and are associated with the BioProject on NCBI (or on FigShare or the AWS database), it is unclear whether the mitochondrial assembly has been made available.
I found that the genome assembly itself on NCBI contains a contig that has been labelled as the mitochondrial genome, JAXCIO010000845.1. However, the length of this sequence (17,265 bp) differs substantially from the mitochondrial genome length reported in the paper (19,498 bp). Is this the same mito assembly described in the paper, or was this contig produced during the de novo assembly of the nuclear genome? I note that the ~17 kbp sequence included in your genome file is more typical for the length of vertebrate mitochondria and is much closer in size to the related parrot you indicated in the paper (Rhynchopsitta pachyrhyncha, OR209192.1) than the ~19kb produced by MitoHifi v2 and reported in-text. I request the following:

If the mitochondrial contig JAXCIO010000845.1 included in the assembly file is the same mitochondrial assembly described in the report, then the length discrepancy between this contig and what is reported in the paper needs to be addressed.
If JAXCIO010000845.1 is not the same as the mito assembly reported in the paper, then 1) your de novo ~19kb mitochondrial genome needs to be deposited into Genbank and associated with your BioProject and 2) the length discrepancy between these two should still be addressed,

"and the impacts of an introduced predator" - it is probably worth spelling out what the predator is.

"to generate a high-quality reference assembly" - . Chromosome-level de novo genome assemblies have become standard, even for vertebrates, and are very slowly starting to be superseded by T2T assemblies. Given current standards and the recent VGP L. discolor reference genome, the assembly presented here would best be described a draft assembly throughout.

Genome assembly methods - What Hifiasm parameters were used? If default that's fine, just note it.

Table 3 "DNA elements" - The label "DNA elements" is default for RepeatMasker but its a fairly vague label for these elements. It would be helpful to rephrase this as "DNA Transposons" to be more specific/clear about what they are.

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Evolutionary Biology, Developmental Biology, Genomics, Marsupials

CITE

Report a concern

Author Response 27 Aug 2024

Luke Silver, School of Life and Environmental Sciences, The University of Sydney, Sydney, 2006, Australia

27 Aug 2024

Author Response

The mitochondrial contig listed on NCBI is the correct mitochondrial assembly, it appears that MitoHifi annotates the entire contig which was identified to contain mitochondrial genome even if it is ... Continue reading The mitochondrial contig listed on NCBI is the correct mitochondrial assembly, it appears that MitoHifi annotates the entire contig which was identified to contain mitochondrial genome even if it is much larger than the expected size.

I had replaced the identified contig with only the portion of sequence which represented the mito genome in the assembly file, however not the data which was used to annotate and produce figure 1.

I have edited and updated the manuscript to reflect the changes. “The mitochondrial reference sequence for the thick-billed parrot was then, used to search for the swift parrot mitochondrial genome. The identified mitochondrial sequence was then added to the genome assembly and annotated using MITOS v 2.1.7 (Donath et al., 2019) and visualised using Proksee ( Grant et al., 2023).”

I have edited the sentence to name the predator “… and the impacts of an introduced predator, the sugar glider (Petaurus breviceps)

I have replaced high-quality with draft when referring to the genome assembly throughout

I have stated that hifiasm was run with default parameters, “Hifiasm, with default parameters, was run on Galaxy Australia…”

I have relabelled DNA elements as DNA transposons in Table 3
The mitochondrial contig listed on NCBI is the correct mitochondrial assembly, it appears that MitoHifi annotates the entire contig which was identified to contain mitochondrial genome even if it is much larger than the expected size.

I had replaced the identified contig with only the portion of sequence which represented the mito genome in the assembly file, however not the data which was used to annotate and produce figure 1.

I have edited and updated the manuscript to reflect the changes. “The mitochondrial reference sequence for the thick-billed parrot was then, used to search for the swift parrot mitochondrial genome. The identified mitochondrial sequence was then added to the genome assembly and annotated using MITOS v 2.1.7 (Donath et al., 2019) and visualised using Proksee ( Grant et al., 2023).”

I have edited the sentence to name the predator “… and the impacts of an introduced predator, the sugar glider (Petaurus breviceps)

I have replaced high-quality with draft when referring to the genome assembly throughout

I have stated that hifiasm was run with default parameters, “Hifiasm, with default parameters, was run on Galaxy Australia…”

I have relabelled DNA elements as DNA transposons in Table 3
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 27 Aug 2024

Luke Silver, School of Life and Environmental Sciences, The University of Sydney, Sydney, 2006, Australia

27 Aug 2024

Author Response

The mitochondrial contig listed on NCBI is the correct mitochondrial assembly, it appears that MitoHifi annotates the entire contig which was identified to contain mitochondrial genome even if it is ... Continue reading The mitochondrial contig listed on NCBI is the correct mitochondrial assembly, it appears that MitoHifi annotates the entire contig which was identified to contain mitochondrial genome even if it is much larger than the expected size.

I had replaced the identified contig with only the portion of sequence which represented the mito genome in the assembly file, however not the data which was used to annotate and produce figure 1.

I have edited and updated the manuscript to reflect the changes. “The mitochondrial reference sequence for the thick-billed parrot was then, used to search for the swift parrot mitochondrial genome. The identified mitochondrial sequence was then added to the genome assembly and annotated using MITOS v 2.1.7 (Donath et al., 2019) and visualised using Proksee ( Grant et al., 2023).”

I have edited the sentence to name the predator “… and the impacts of an introduced predator, the sugar glider (Petaurus breviceps)

I have replaced high-quality with draft when referring to the genome assembly throughout

I have stated that hifiasm was run with default parameters, “Hifiasm, with default parameters, was run on Galaxy Australia…”

I have relabelled DNA elements as DNA transposons in Table 3
The mitochondrial contig listed on NCBI is the correct mitochondrial assembly, it appears that MitoHifi annotates the entire contig which was identified to contain mitochondrial genome even if it is much larger than the expected size.

I had replaced the identified contig with only the portion of sequence which represented the mito genome in the assembly file, however not the data which was used to annotate and produce figure 1.

I have edited and updated the manuscript to reflect the changes. “The mitochondrial reference sequence for the thick-billed parrot was then, used to search for the swift parrot mitochondrial genome. The identified mitochondrial sequence was then added to the genome assembly and annotated using MITOS v 2.1.7 (Donath et al., 2019) and visualised using Proksee ( Grant et al., 2023).”

I have edited the sentence to name the predator “… and the impacts of an introduced predator, the sugar glider (Petaurus breviceps)

I have replaced high-quality with draft when referring to the genome assembly throughout

I have stated that hifiasm was run with default parameters, “Hifiasm, with default parameters, was run on Galaxy Australia…”

I have relabelled DNA elements as DNA transposons in Table 3
Competing Interests: No competing interests were disclosed. Close
Report a concern

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 04 Apr 2024

Open Peer Review

Reviewer Status

Reviewer Reports

	Invited Reviewers
	1	2	3
Version 2 (revision) 27 Aug 24		read	read
Version 1 04 Apr 24	read	read

Charles Feigin, La Trobe University (Ringgold ID: 2080), Melbourne, Australia
Phred Benham, University of Massachusetts Amherst Department of Biology (Ringgold ID: 117236), Amherst, USA
Joan Ferrer Obiol, University of Milan, Milan, Italy; Universitat de Barcelona (UB), Barcelona, Spain

Comments on this article

All Comments(0)

Add a comment

Browse by related subjects

Back to all reports

Reviewer Report

6 Views

18 Sep 2024 | for Version 2

Joan Ferrer Obiol, University of Milan, Milan, Italy; Universitat de Barcelona (UB), Barcelona, Spain

6 Views Cite this report Responses(0)

Approved

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

References

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Population genomics, phylogenomics, ecological genomics and some experience in genome assembly

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

4 Views

10 Sep 2024 | for Version 2

Phred Benham, University of Massachusetts Amherst Department of Biology (Ringgold ID: 117236), Amherst, Massachusetts, USA

4 Views Cite this report Responses(0)

Approved

The authors have addressed my comments and those of the other reviewer. I have no further comments to add.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

genomics, ornithology, evolution

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

8 Views

13 Jul 2024 | for Version 1

Phred Benham, University of Massachusetts Amherst Department of Biology (Ringgold ID: 117236), Amherst, Massachusetts, USA

8 Views Cite this report Responses(1)

Approved With Reservations

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

genomics, ornithology, evolution

Respond to this report

Responses (1)

Author Response

27 Aug 2024

Luke Silver, School of Life and Environmental Sciences, The University of Sydney, Sydney, 2006, Australia

We did not attempt a scaffolded assembly as for conservation and population genetics work we have found a long read assembly to be sufficient, additionally after we produced our long read assembly a chromosome level scaffolded assembly was released by VGP.

In lieu of adding in an additional figure showing the distribution of the swift parrot, we have added some additional detail as to the regions where swift parrots breed and migrate to
“The swift parrot ( Lathamus discolor) is a migratory parrot that breeds on the eastern seaboard of the island of Tasmania, Australia and winters on southeastern mainland Australia”

As requested by the other reviewer I have ensured the mitochondrial information included in the manuscript and online is correct and updated to reflect this.

The individual used for the genome and transcriptome will be accessioned into a museum collection by the end of 2024, when this has been completed the BioProject information on NCBI will be updated to provide the accession number

I have rephrased this to make sense. “…genome as, the thick-billed parrot ( Rhynchopsitta pachyrhyncha) (NCBI reference sequence OR209192.1). The mitochondrial reference sequence for the thick-billed parrot was then, used…”

I have changed with to were

The crow is the most closely related species with a matrix provided by FGENESH, I have edited the methods to reflect this. “…gene finding matrix, which is the closest related species with a gene finding matrix provided by FGENESH++”

View more View less

Competing Interests

No competing interests were disclosed.

Back to all reports

Reviewer Report

13 Views

04 Jun 2024 | for Version 1

Charles Feigin, Department of Environment and Genetics, La Trobe University (Ringgold ID: 2080), Melbourne, Victoria, Australia

13 Views Cite this report Responses(1)

Approved With Reservations

If the mitochondrial contig JAXCIO010000845.1 included in the assembly file is the same mitochondrial assembly described in the report, then the length discrepancy between this contig and what is reported in the paper needs to be addressed.
If JAXCIO010000845.1 is not the same as the mito assembly reported in the paper, then 1) your de novo ~19kb mitochondrial genome needs to be deposited into Genbank and associated with your BioProject and 2) the length discrepancy between these two should still be addressed,

Are the rationale for sequencing the genome and the species significance clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Evolutionary Biology, Developmental Biology, Genomics, Marsupials

Respond to this report

Responses (1)

Author Response

27 Aug 2024

Luke Silver, School of Life and Environmental Sciences, The University of Sydney, Sydney, 2006, Australia

The mitochondrial contig listed on NCBI is the correct mitochondrial assembly, it appears that MitoHifi annotates the entire contig which was identified to contain mitochondrial genome even if it is much larger than the expected size.

I had replaced the identified contig with only the portion of sequence which represented the mito genome in the assembly file, however not the data which was used to annotate and produce figure 1.

I have edited and updated the manuscript to reflect the changes. “The mitochondrial reference sequence for the thick-billed parrot was then, used to search for the swift parrot mitochondrial genome. The identified mitochondrial sequence was then added to the genome assembly and annotated using MITOS v 2.1.7 (Donath et al., 2019) and visualised using Proksee ( Grant et al., 2023).”

I have edited the sentence to name the predator “… and the impacts of an introduced predator, the sugar glider (Petaurus breviceps)

I have replaced high-quality with draft when referring to the genome assembly throughout

I have stated that hifiasm was run with default parameters, “Hifiasm, with default parameters, was run on Galaxy Australia…”

I have relabelled DNA elements as DNA transposons in Table 3

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] Afgan E, Baker D, van den Beek M , et al.: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 2016; 44(W1): W3–W10. PubMed Abstract | Publisher Full Text | Free Full Text

[2] Allio R, Schomaker-Bastos A, Romiguier J, et al.: MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics. Mol. Ecol. Resour. 2020; 20(4): 892–905. PubMed Abstract | Publisher Full Text | Free Full Text

[3] BirdLife International: Lathamus discolor. The IUCN Red List of Threatened Species 2018.2018. Publisher Full Text

[4] Bolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract | Publisher Full Text | Free Full Text

[5] Cheng H, Jarvis ED, Fedrigo O, et al.: Haplotype-resolved assembly of diploid genomes without parental data. Nat. Biotechnol. 2022; 40(9): 1332–1335. PubMed Abstract | Publisher Full Text | Free Full Text

[6] Danecek P, Bonfield JK, Liddle J, et al.: Twelve years of SAMtools and BCFtools. Gigascience. 2021; 10(2). PubMed Abstract | Publisher Full Text | Free Full Text

[7] Donath A, Jühling F, Al-Arab M, et al.: Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Res. 2019 Nov 18; 47(20): 10543–10552. PubMed Abstract | Publisher Full Text | Free Full Text

[8] Flynn JM, Hubley R, Goubert C, et al.: RepeatModeler2 for automated genomic discovery of transposable element families. Proc. Natl. Acad. Sci. USA. 2020; 117(17): 9451–9457. PubMed Abstract | Publisher Full Text | Free Full Text

[9] Grant JR, Enns E, Marinier E, et al.: Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023; 51(W1): W484–W492. PubMed Abstract | Publisher Full Text | Free Full Text

[10] Heinsohn R, Webb M, Lacy R, et al.: A severe predator-induced population decline predicted for endangered, migratory swift parrots (Lathamus discolor). Biol. Conserv. 2015; 186: 75–82. Publisher Full Text

[11] Kang YJ, Yang DC, Kong L, et al.: CPC2: a fast and accurate coding potential calculator based on sequence intrinsic features. Nucleic Acids Res. 2017; 45(W1): W12–W16. PubMed Abstract | Publisher Full Text | Free Full Text

[12] Kennedy SJ, Tzaros CL: Foraging ecology of the swift parrot Lathamus discolor in the box-ironbark forests and woodlands of Victoria. Pac. Conserv. Biol. 2005; 11(3): 158–173. Publisher Full Text

[13] Kim D, Paggi JM, Park C, et al.: Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 2019; 37(8): 907–915. PubMed Abstract | Publisher Full Text | Free Full Text

[14] Li H, Handsaker B, Wysoker A, et al.: The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. PubMed Abstract | Publisher Full Text | Free Full Text

[15] MacNally R, Horrocks G: Landscape-scale conservation of an endangered migrant:the swift parrot (Lathamus discolor) in its winter range. Biol. Conserv. 2000; 92(3): 335–343. Publisher Full Text

[16] Morrison CE, Hogg CJ, Gales R, et al.: Low innate immune-gene diversity in the critically endangered orange-bellied parrot (Neophema chrysogaster). Emu Austral Ornithol. 2020; 120(1): 56–64. Publisher Full Text

[17] Olah G, Stojanovic D, Webb MH, et al.: Comparison of three techniques for genetic estimation of effective population size in a critically endangered parrot. Anim. Conserv. 2021; 24(3): 491–498. Publisher Full Text

[18] Owens G, Heinsohn R, Crates R, et al.: Long-term ecological data confirm and refine conservation assessment of critically endangered swift parrots. Anim. Conserv. 2023; 26(4): 450–463. Publisher Full Text

[19] Pertea M, Pertea GM, Antonescu CM, et al.: StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 2015; 33(3): 290–295. PubMed Abstract | Publisher Full Text | Free Full Text

[20] Price G, Farquharson K: PacBio HiFi genome assembly using hifiasm v2.1. WorkflowHub. 2022. Publisher Full Text

[21] Saunders DL, Heinsohn R: Winter habitat use by the endangered, migratory swift parrot (Lathamus discolor) in New South Wales. Emu Austral Ornithol. 2008; 108(1): 81–89. Publisher Full Text

[22] Saunders DL, Tzaros CL: National recovery plan for the swift parrot Lathamus discolor. Melbourne: Birds Australia; 2011.

[23] Silver LW, Stojanovic D, Farquharson K, et al.: Lathamus discolor Genome sequencing and assembly.2023. Reference Source

[24] Simao FA, Waterhouse RM, Ioannidis P, et al.: BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics. 2015; 31(19): 3210–3212. Publisher Full Text

[25] Smit A, Hubley R, Green P: RepeatMasker Open-4.0.2013-2015.

[26] Solovyev V, Kosarev P, Seledsov I, et al.: Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biol. 2006; 7 Suppl 1(Suppl 1): S10.1–S1012. PubMed Abstract | Publisher Full Text | Free Full Text

[27] Stojanovic D, Olah G, Webb M, et al.: Genetic evidence confirms severe extinction risk for critically endangered swift parrots: Implications for conservation management. Anim. Conserv. 2018; 21(4): 313–323. Publisher Full Text

[28] Uliano-Silva M, Ferreira JGRN, Krasheninnikova K, et al.: MitoHiFi: a python pipeline for mitochondrial genome assembly from PacBio High Fidelity reads. bioRxiv. 2023. 2022.2012.2023.521667. Publisher Full Text

[29] Webb MH, Stojanovic D, Heinsohn R: Policy failure and conservation paralysis for the critically endangered swift parrot. Pac. Conserv. Biol. 2019; 25(2): 116–123. Publisher Full Text

[30] Zhang G, Li C, Li Q, et al.: Comparative genomics reveals insights into avian genome evolution and adaptation. Science. 2014; 346(6215): 1311–1320. PubMed Abstract | Publisher Full Text | Free Full Text

A reference genome, mitochondrial genome and associated transcriptomes for the critically endangered swift parrot (Lathamus discolor)

Abstract

Abstract*

Keywords

Revised Amendments from Version 1

Introduction

Methods

Sample collection and DNA/RNA extraction

Library construction and sequencing

Genome assembly

Mitochondrial assembly

Transcriptome assembly

Genome annotation

Results

Genome assembly

Figure 1. Mitochondrial genome of the swift parrot (Lathamus discolor) generated with Proksee (Grant et al., 2023).

Transcriptome assembly and genome annotation

Table 2. Statistics of the global transcriptome and annotation of the swift parrot (Lathamus discolor) including BUSCO (Simao et al., 2015) completeness, calculated with both the vertebrata_obd10 and aves_obd10 lineages and average exon length.

Table 3. Classification of repeat elements of the swift parrot (Lathamus discolor) genome assembly as generated by the repeatmasker software (Smit et al., 2013-2015).

Ethical considerations

Data availability

Underlying data

Reporting guidelines

Acknowledgements

References

Comments on this article Comments (0)

Open Peer Review

Comments on this article Comments (0)

Open Peer Review

Reviewer Status

Reviewer Reports

Comments on this article

Browse by related subjects

Competing Interests Policy

Stay Updated