Abi 3730xl dna analyser

Total DNA was extracted from blood samples using a DNeasy Blood and tissue kit (Qiagen, UK). Mitochondrial genes (Cyt b, ND3) and a nuclear gene (TGFß2) were selected based on their performance from previous Zosterops studies (Warren et al. 2006 (link); Moyle et al. 2009 ; Melo et al. 2011 (link)). Amplification of ND3 and TGFß2 was performed using published primers (Table S2, Supporting information). To obtain the entire Cyt b gene, the published primer H16065 was used alongside three newly designed primers (Table S2, Supporting information). PCR amplifications and thermal cycling conditions for all three genes are reported in Table S2. Cleaned products were sequenced on an ABI 3730xl DNA analyser (Applied Biosystems, UK).

PCR products were sequenced via Sanger sequencing using a capillary ABI 3730xl DNA Analyser (Applied Biosystems) at Genetic Analysis Services (GAS), Department of Anatomy, University of Otago (New Zealand). Geneious^® (Geneious Pro software, Biomatters Ltd., Auckland, New Zealand) version 10.2.3 was used to analyse sequencing data and investigate/compare genome sequences between species to carry out sequence analysis. For analyses involving the Medicago genome, the latest version of the Medicago truncatula genome (Medtr 4.0v2) with annotations was used as a reference [34 (link)]. Protein alignment was performed using Geneious^® MUSCLE Alignment with default settings and the domain search was performed in http://www.ebi.ac.uk/interpro/ (accessed on 6 December 2021) Fe_bilin_red domain (from 63aa to 280aa).

DNA was extracted using an Ezup Column Animal Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) following the manufacturer’s standard protocols. Primers used were LCO1490/HCO2198, which are commonly used for metazoans (Folmer et al. 1994 (link)). PCR amplification of mitochondrial COI was performed in 25 μL volumes containing 12.5 μL of Premix Taq (TaKaRa Taq v. 2.0 plus dye), 1.25 μL of each primer, 8 μL of ddH₂O, 2 μL of template DNA, with PCR programs following Zhang et al. (2014) (link). All PCR products were checked on a 1% agarose gel. Successful products were purified and sequenced in both directions by Majorbio (Shanghai, China) on an ABI 3730XL DNA Analyser (Applied Biosystems). COI sequences for the remaining species were obtained from the NCBI (https://www.ncbi.nlm.nih.gov/). Sequences were preliminarily aligned using MAFFT v. 7.450 by the L-INS-I strategy (Katoh and Standley 2013 (link)) and corrected manually, with a final 658-bp alignment. Neighbour-joining (NJ) tree and Kimura 2-parameter (K2P; Kimura 1980 (link)) distances were calculated in MEGA v. 7.0 (Kumar et al. 2016 (link)). Node supports were evaluated through 1,000 bootstrap replications.

DNA was extracted using a QIAamp DNA Micro Kit (QIAGEN GmbH, Shanghai, China). Extractions were performed non-destructively for further morphological examination and identification of the specimens. DNA concentration was measured by Qubit 3.0 using Q33230 Qubit™ 1X dsDNA HS Assay Kit. Mitogenome amplification for less than 50 ng DNA was performed using REPLI-g Single Cell Kit. Each library was sequenced with an insert size of 350 bp on HiSeq X Ten platform (Tianjin Novogene Bioinformatics Technology Co., Ltd, China) generating 150 bp paired-end reads.
The COX1 was used as a seed sequence for our mitochondrial assembly. Primers for COX1 were LCO1490/HCO2198 commonly used for Metazoa [45 (link)]. Amplification volume and procedure followed Zhang et al. [46 ]. All PCR products were checked on a 1% agarose gel. Successful products were purified and sequenced in both directions by Tsingke (Beijing, China) on ABI 3730XL DNA Analyser (Applied Biosystems). Sequences were assembled in Sequencher 4.5 (Gene Codes Corporation, Ann Arbor, USA), blasted in GenBank and checked for possible errors, then were preliminarily aligned using MEGA 7.0 [47 (link)]. Alignments were checked and corrected manually, with a final 658 bps alignment.

DNA sequencing was performed using an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) in the paired-end mode using a v3 chemistry kit (Illumina). The obtained sequence reads were filtered for quality using fastp v0.19.5 with a window size of 10 bps moving from 5′ to 3′ and removing bps with a quality lower than 15, polyX at the 3′ end, and the reads of lengths lower than 150 bps [67 (link)]. Afterwards, the reads were assembled using SPAdes v3.11.1 with default parameters [68 (link)]. The presence of redundant ends in the assembled contigs was verified by remapping the reads against the assembled genomes using bwa mem v0.7.17-r1188, processed afterwards with samtools v1.7 and viewed in the Integrative Genome Viewer v2.6.2 [69 ,70 (link),71 (link)]. Circulation of the genomes was validated by capillary sequencing of the PCR products using an ABI3730xl DNA Analyser (Applied Biosystems, Waltham, MA, USA).

The mtDNA control region analyses showed the presence of two major clades, a small clade separated from a large clade without any geographical pattern (Figure 1). To understand this result and determine if it is, due to the presence of nuclear copies of mitochondrial genomes or indicative of introgression, the cytochrome b gene was examined in a selection of seven samples; four from the small clade and three from the large clade (detailed methods are outlined in Supplementary Material, Text S2). A ~1000 bp fragment containing the entire cytochrome b gene was amplified using PCR primers H16064 (5’-CTTCAGTTTTTGGTTTACAAGACC-3’) and L14764 (5’-TGRTACAAAAAAATAGGMCCMGAAGG-3’) [33 (link)]. Purified PCR products (Acroprep 96 filter plates, PALL Corporation, Port Washington, NY, USA) were sequenced (BigDye Terminator v.3.1, ThermoFisher, Waltham, MA, USA) on an ABI 3730xl DNA analyser (Applied Biosystems Inc., Foster City, CA, USA) with one primer, L14764.
Sequences were aligned in Geneious^® 9.0.5 [33 (link)]. Sequences for all seven samples were aligned with a 1143 bp cytochrome b sequence of the red-billed gull (NCBI Accession No.: FM209918) and a 1143 bp cytochrome b sequence of a black-billed gull (NCBI Accession No.: FM209900.1 used as a comparison to our sequences) to determine the presence of introgression. This alignment was used to produce a neighbor-joining tree in Geneious^®.

DNA was extracted from the 436 K. pneumoniae strains using the QIAamp DNA mini kit (QIAGEN, Düsseldorf, Germany) according to the manufacturer’s protocol. Seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) were amplified via polymerase chain reaction (PCR) (Diancourt et al., 2005 (link)) and then sequenced using an ABI 3730XL DNA Analyser (Applied Biosystems, San Ramon, CA, USA), and then compared with sequences available on the K. pneumoniae MLST database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). The primers used are shown in Table S1.