To export or not export phruta outputs

To export or not export from phruta
From taxonomic names to sequence alignments, exporting

To export or not export from `phruta`

Assembling a molecular dataset for particular target taxa with phruta can be performed almost entirely by saving objects into your workspace. This topic was covered in the introductory vignette to phruta (Using the phruta R package). However, in some situations, it might be desirable to save the outputs of different phruta functions to particular folders. This tutorial will cover that specific situation. Specifically, we will be reviewing how phruta can be used to export .fasta and .csv files that are generated in different steps of the pipeline.

From taxonomic names to sequence alignments, exporting data

In the introductory vignette to phruta (Using the phruta R package), we assembled a basic molecular dataset for inferring the phylogeny among three mammal genera. Let’s recreate the same tutorial, but in this case, exporting the intermediate files that are created after using several of the functions. Note that the structure of this tutorial closely follows that of “Using the phruta R package”. You should be able to follow this tutorial even without having reviewed the introductory vignette.

Please assume that we are interested in building a phylogenetic tree for the following three genera: Felis, Vulpes, and Phoca. All these three genera are classified within the Carnivora, a mammalian order. Both Felis and Vulpes are classified in different superfamilies within the Fissipedia. Finally, Phoca is part of another suborder, Pinnipedia. We’re going to root our tree with another mammal species, a Chinese Pangolin (Manis pentadactyla). Users can select additional target species and clades. However, for simplicity, we will run the analyses using three genera in the ingroup and a single outgroup species.

So far, we have decided the taxonomic make of our analyses in phruta. We will also need to determine the gene regions to be used in our analyses. Fortunately, mammals are extensively studied and a comprehensive list of potential gene regions to be analyzed is already available. For instance, we could use same gene regions sampled in Upham et al (2009). However, for this tutorial, we will simply try to find the gene regions are well sampled for the target taxa. I believe that figuring out the best sampled gene regions in genbank, instead of providing gene names, is potentially more valuable when working with poorly studied groups (e.g. invertebrates). Before we move on, please make sure that you you have set a working directory for this project. All the files will be saved to this directory. phruta will politely ask before writing files to your local directories.

Let’s start by loading phruta!

library(phruta)

Now, let’s look for the gene regions that are sampled for our target taxa. Again, this step is not always necessary. In some groups, gene-level sampling is very standard (e.g. COI, 12S). However, the structure of gene sampling sometimes becomes more blurry as you zoom out taxonomically. For instance, genes A and B can be extensively sampled in genus 1. However, genus 2 in the same family has mainly been studied using genes Y and Z. The idea here is that phruta will try to find those gene regions that are extensively sampled across species in the target taxa. We will use the gene.sampling.retrieve() function in phruta. The resulting data.frame, named gs.seqs in this example, will contain the list of full names for genes sampled in genbank for the target taxa.

gs.seqs <- gene.sampling.retrieve(organism = c("Felis", "Vulpes", "Phoca", "Manis_pentadactyla"), 
                                  speciesSampling = TRUE)

For the search terms used above, phruta was able to retrieve the names for 1594 gene regions. In the table below I summarize a few of those genes, with sampling frequency calculated at the level of species (see speciesSampling = TRUE argument above).

Gene sampling for the search including the genera Felis, Vulpes, Phoca as the ingroup taxa. Manis pentadactyla was used as outgroup.
Gene	Sampled in N species	PercentOfSampledSpecies
cytochrome b	25	75.75758
NADH dehydrogenase subunit 5	14	42.42424
12S ribosomal RNA	11	33.33333
cytochrome oxidase subunit 1	11	33.33333
growth hormone receptor	11	33.33333
interphotoreceptor retinoid-binding protein	10	30.30303

Thus, the gene.sampling.retrieve() function provides an estimate of the number of species in genbank that matches the taxonomic criteria and have sequences for a given gene region. Note that the estimates recovered by gene.sampling.retrieve() are only as good as the annotations that other researchers have provided for sequences deposited in genbank.

From here, we will generate a preliminary table summarizing accession numbers for the combination of taxa and gene regions that we’re interested in sampling. However, note that not all these accession numbers are expected to be in the final (curated) molecular dataset. For instance, several sequences might be dropped later after taxonomic information is curated. Now, we will assemble a species-level summary of accession numbers using the acc.table.retrieve() function. For simplicity, this tutorial will focus on sampling gene regions that are sampled in >30% of the species (targetGenes data.frame).

targetGenes <- gs.seqs[gs.seqs$PercentOfSampledSpecies > 30,]

acc.table <- acc.table.retrieve(
            clades  = c('Felis', 'Vulpes', 'Phoca'),
            species = 'Manis_pentadactyla' ,
            genes   = targetGenes$Gene,
            speciesLevel = TRUE
          )

The acc.table object is a data.frame that will be used below for downloading the relevant gene sequences. In this case, the dataset includes the following information:

Accession numbers for the relevant species and gene regions examined in this tutorial.
Species	Ti	Acc	gene
Felis silvestris	Felis silvestris silvestris isolate FS_101 NADH dehydrogenase subunit 5 (ND5) gene, partial cds; NADH dehydrogenase subunit 6 (ND6) gene, complete cds; tRNA-Glu gene, complete sequence; and cytochrome b (cytb) gene, partial cds; mitochondrial	OL654361	cytochrome b
Felis catus	Felis catus MKRaS008 mitochondrial gene for cytochrome b, partial cds	LC649705	cytochrome b
Felis chaus	Felis chaus isolate Jungle Cat 5 cytochrome b (cytb) gene, partial cds; mitochondrial	MN370575	cytochrome b
Felis environmental	Felis environmental sample isolate Kw-170 cytochrome b (cytb) gene, partial cds; mitochondrial	MK510873	cytochrome b
Felis margarita	Felis margarita haplotype AH NADH dehydrogenase subunit 5 (ND5) and cytochrome b (cytb) genes, partial cds; and tRNA-Thr gene and D-loop, partial sequence; mitochondrial	MK606132	cytochrome b
Felis bieti	Felis bieti cytochrome b gene, partial cds; mitochondrial	AY773081	cytochrome b
F.domesticus mitochondrial	F.domesticus mitochondrial cytochrome b gene	X82296	cytochrome b
Vulpes vulpes	Vulpes vulpes isolate LH198 haplotype FOX14 cytochrome b (CYTB) gene, partial cds; mitochondrial	MK244493	cytochrome b
Vulpes corsac	Vulpes corsac isolate SH21 cytochrome b (CYTB) gene, complete cds; mitochondrial	MT795179	cytochrome b
Vulpes zerda	Vulpes zerda isolate X161349 cytochrome b (Cytb) gene, partial cds; mitochondrial	MH854561	cytochrome b
Vulpes cana	Vulpes cana isolate B.F.Y3 cytochrome b (cytb) gene, partial cds; mitochondrial	KU378587	cytochrome b
Vulpes rueppellii	Vulpes rueppellii isolate R.F.Y6 cytochrome b (cytb) gene, partial cds; mitochondrial	KU378373	cytochrome b
Vulpes lagopus	Vulpes lagopus haplotype 5 cytochrome b (cytb) gene, partial cds; mitochondrial	KX093945	cytochrome b
Vulpes ferrilata	Vulpes ferrilata haplotype 1 cytochrome b (cytb) gene, partial cds; mitochondrial	EU872065	cytochrome b
Vulpes macrotis	Vulpes macrotis cytochrome b (cytb) gene, mitochondrial gene encoding mitochondrial protein, partial cds	AF028157	cytochrome b
Vulpes pallida	Vulpes pallida haplotype PMa2 cytochrome b (cytb) gene, partial cds; mitochondrial	KJ597964	cytochrome b
V.vulpes mitochondrial	V.vulpes mitochondrial DNA for cytochrome b (complete sequence)	X94929	cytochrome b
Phoca largha	Phoca largha PLCBRe4 mitochondrial cytb gene for cytochrome b, partial cds	LC466149	cytochrome b
Pagophilus groenlandicus	Pagophilus groenlandicus cytochrome b gene, partial cds; mitochondrial gene for mitochondrial product	AF200491	cytochrome b
Phoca groenlandica	Phoca groenlandica cytochrome b (cytb) gene, complete cds; mitochondrial	GU174609	cytochrome b
Phoca fasciata	Phoca fasciata cytochrome b (cytb) gene, complete cds; mitochondrial	GU167294	cytochrome b
Phoca vitulina	Phoca vitulina mitochondrial cytochrome b gene, partial cds	L19127	cytochrome b
P.vitulina mitochondrial	P.vitulina mitochondrial cytochrome b gene	X82306	cytochrome b
P.largha mitochondrial	P.largha mitochondrial cytochrome b gene	X82305	cytochrome b
P.groenlandica mitochondrial	P.groenlandica mitochondrial cytochrome b gene	X82303	cytochrome b
P.fasciata mitochondrial	P.fasciata mitochondrial cytochrome b gene	X82302	cytochrome b
Manis pentadactyla	Manis pentadactyla isolate ST08 cytochrome b (cytb) gene, partial cds; mitochondrial	MW197469	cytochrome b
Felis silvestris	Felis silvestris silvestris isolate FS_101 NADH dehydrogenase subunit 5 (ND5) gene, partial cds; NADH dehydrogenase subunit 6 (ND6) gene, complete cds; tRNA-Glu gene, complete sequence; and cytochrome b (cytb) gene, partial cds; mitochondrial	OL654361	NADH dehydrogenase subunit 5
Felis catus	Felis catus isolate 4709-K NADH dehydrogenase subunit 5 (ND5) gene, partial cds; NADH dehydrogenase subunit 6 (ND6) gene, complete cds; tRNA-Glu gene, complete sequence; and cytochrome b (CYTB) gene, partial cds; mitochondrial	MN313781	NADH dehydrogenase subunit 5
Felis margarita	Felis margarita haplotype AH NADH dehydrogenase subunit 5 (ND5) and cytochrome b (cytb) genes, partial cds; and tRNA-Thr gene and D-loop, partial sequence; mitochondrial	MK606132	NADH dehydrogenase subunit 5
Felis chaus	Felis chaus isolate JCAIZ003 NADH dehydrogenase subunit 5 (ND5) gene, partial cds; mitochondrial	GU561700	NADH dehydrogenase subunit 5
Vulpes lagopus	Vulpes lagopus ATP synthase F0 subunit 6 (ATP6), ATP synthase F0 subunit 8 (ATP8), cytochrome c oxidase subunit I (COX1), cytochrome c oxidase subunit II (COX2), cytochrome c oxidase subunit III (COX3), cytochrome b (CYTB), NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 2 (ND2), NADH dehydrogenase subunit 3 (ND3), NADH dehydrogenase subunit 4 (ND4), NADH dehydrogenase subunit 4L (ND4L), and NADH dehydrogenase subunit 5 (ND5) genes, complete cds; mitochondrial	AH014073	NADH dehydrogenase subunit 5
Phoca groenlandica	Phoca groenlandica NADH dehydrogenase subunit 5 (ND5) gene, complete cds; mitochondrial gene for mitochondrial product	AY377376	NADH dehydrogenase subunit 5
Phoca fasciata	Phoca fasciata NADH dehydrogenase subunit 5 (ND5) gene, complete cds; mitochondrial	GU167331	NADH dehydrogenase subunit 5
Felis catus	Felis catus voucher N22b 12S ribosomal RNA gene, partial sequence; mitochondrial	KX786344	12S ribosomal RNA
Felis chaus	Felis chaus isolate G 12S ribosomal RNA gene, partial sequence; mitochondrial	KU963205	12S ribosomal RNA
Felis silvestris	Felis silvestris 12S ribosomal RNA gene, partial sequence; mitochondrial	KX002032	12S ribosomal RNA
Felis bieti	Felis bieti 12S ribosomal RNA gene, partial sequence; mitochondrial	AY773084	12S ribosomal RNA
Vulpes vulpes	Vulpes vulpes Vv1 mitochondrial gene for 12S ribosomal RNA, partial sequence	LC424764	12S ribosomal RNA
Vulpes lagopus	Vulpes lagopus isolate FRT12 12S ribosomal RNA gene, partial sequence; mitochondrial	KM224240	12S ribosomal RNA
Phoca fasciata	Phoca fasciata isolate 5888 12S ribosomal RNA gene, partial sequence; mitochondrial	GU174595	12S ribosomal RNA
Phoca largha	Phoca largha isolate 06spotted03 12S ribosomal RNA gene, partial sequence; mitochondrial	GU174591	12S ribosomal RNA
Manis pentadactyla	Manis pentadactyla 12S ribosomal RNA gene, partial sequence; and tRNA-Val gene, complete sequence; mitochondrial	AY012154	12S ribosomal RNA
Felis catus	Felis catus voucher Cat_KU cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	MN124254	cytochrome oxidase subunit 1
Felis nigripes	Felis nigripes voucher NZG:BWP38761 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	KX012677	cytochrome oxidase subunit 1
Felis margarita	Felis margarita voucher 198_Fe_mar cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	KF297765	cytochrome oxidase subunit 1
Vulpes vulpes	Vulpes vulpes voucher BIOUG:HBL008452 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443560	cytochrome oxidase subunit 1
Vulpes chama	Vulpes chama voucher NZG:BWP38701 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	KX012672	cytochrome oxidase subunit 1
Vulpes lagopus	Vulpes lagopus voucher HBL008485 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443554	cytochrome oxidase subunit 1
Vulpes velox	Vulpes velox voucher ROM 105399 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443557	cytochrome oxidase subunit 1
Phoca vitulina	Phoca vitulina voucher HBL008389 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443364	cytochrome oxidase subunit 1
Phoca largha	Phoca largha voucher HBL008423 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443363	cytochrome oxidase subunit 1
Phoca groenlandica	Phoca groenlandica voucher HBL008364 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443362	cytochrome oxidase subunit 1
Manis pentadactyla	Manis pentadactyla isolate KFBG_HZ0050 cytochrome oxidase subunit 1 gene, partial cds; mitochondrial	KT428152	cytochrome oxidase subunit 1
Felis catus	Felis catus growth hormone receptor (GHR) gene, partial cds	DQ205829	growth hormone receptor
Vulpes vulpes	Vulpes vulpes growth hormone receptor gene, exon 10 and partial cds	AY885401	growth hormone receptor
Vulpes macrotis	Vulpes macrotis growth hormone receptor gene, exon 10 and partial cds	AY885400	growth hormone receptor
Vulpes corsac	Vulpes corsac growth hormone receptor gene, exon 10 and partial cds	AY885399	growth hormone receptor
Vulpes zerda	Vulpes zerda growth hormone receptor gene, exon 10 and partial cds	AY885393	growth hormone receptor
Alopex lagopus	Alopex lagopus growth hormone receptor gene, exon 10 and partial cds	AY885379	growth hormone receptor
Vulpes velox	Vulpes velox growth hormone receptor (GHR) gene, partial cds	DQ205838	growth hormone receptor
Vulpes lagopus	Vulpes lagopus growth hormone receptor (GHR) gene, partial cds	DQ205837	growth hormone receptor
Phoca vitulina	Phoca vitulina growth hormone receptor (GHR) gene, partial cds	GU931127	growth hormone receptor
Phoca largha	Phoca largha growth hormone receptor (GHR) gene, partial cds	DQ205827	growth hormone receptor
Phoca groenlandica	Phoca groenlandica growth hormone receptor (GHR) gene, partial cds	DQ205825	growth hormone receptor
Manis pentadactyla	Manis pentadactyla growth hormone receptor (GHR) gene, exon 10 and partial cds	EU448992	growth hormone receptor
Felis catus	Felis catus interphotoreceptor retinoid binding protein gene, exon 1	Z11811	interphotoreceptor retinoid-binding protein
Vulpes velox	Vulpes velox interphotoreceptor retinoid binding protein gene, partial cds	AF179293	interphotoreceptor retinoid-binding protein
Manis pentadactyla	Manis pentadactyla interphotoreceptor retinoid binding protein (IRBP) gene, exon 1 and partial cds	JN414784	interphotoreceptor retinoid-binding protein
Felis silvestris	Felis silvestris haplotype W23 tRNA-Pro gene and control region, partial sequence; mitochondrial	MF353436	tRNA-Pro
Felis catus	Felis catus mitochondrial tRNA-Pro gene and control region, partial sequence	AF348642	tRNA-Pro
Vulpes vulpes	Vulpes vulpes isolate VvUAE056 tRNA-Thr gene, partial sequence; tRNA-Pro gene, complete sequence; and D-loop, partial sequence; mitochondrial	MT955893	tRNA-Pro
Vulpes rueppellii	Vulpes rueppellii isolate VrKSAA0005 tRNA-Thr gene, partial sequence; tRNA-Pro gene, complete sequence; and D-loop, partial sequence; mitochondrial	MT955814	tRNA-Pro
Vulpes lagopus	Vulpes lagopus haplotype H11 tRNA-Pro gene and D-loop, partial sequence; mitochondrial	KX093931	tRNA-Pro
Vulpes macrotis	Vulpes macrotis haplotype Vmac1 tRNA-Pro gene and D-loop, partial sequence; mitochondrial	KJ846673	tRNA-Pro
Vulpes zerda	Vulpes zerda haplotype Vzer1 tRNA-Pro gene and D-loop, partial sequence; mitochondrial	KJ846672	tRNA-Pro
Vulpes ferrilata	Vulpes ferrilata haplotype 4 D-loop, partial sequence; tRNA-Pro gene, complete sequence; and tRNA-Thr gene, partial sequence; mitochondrial	JF520840	tRNA-Pro
Phoca largha	Phoca largha isolate pl27 tRNA-Thr gene, partial sequence; tRNA-Pro gene, complete sequence; and D-loop, partial sequence; mitochondrial	OM967017	tRNA-Pro
Phoca vitulina	Phoca vitulina isolate N tRNA-Thr (trnT) gene, partial sequence; tRNA-Pro (trnP) gene, complete sequence; and D-loop, partial sequence; mitochondrial	HQ702987	tRNA-Pro
Manis pentadactyla	Manis pentadactyla pentadactyla isolate MPP5 tRNA-Pro gene, partial sequence; D-loop, complete sequence; and tRNA-Phe gene, partial sequence; mitochondrial	GQ232081	tRNA-Pro

Feel free to review this dataset, make changes, add new species, samples, etc. The integrity of this dataset is critical for the next steps so please take your time and review it carefully. For instance, let’s just make some minor changes to our dataset:

acc.table$Species <- sub("P.", "Phoca ", acc.table$Species, fixed = TRUE)
acc.table$Species <- sub("F.", "Felis ", acc.table$Species, fixed = TRUE)
acc.table$Species <- sub("V.", "Vulpes ", acc.table$Species, fixed = TRUE)
acc.table$Species <- sub("mitochondrial", "", acc.table$Species)
row.names(acc.table) <- NULL

Let’s check how the new table looks now…

Updated Table 2 with some minor corrections on species names. Other updates to this table include adding more sequences, using specific sequences (instead of the ones suggested by `phruta`).
Species	Ti	Acc	gene
Felis silvestris	Felis silvestris silvestris isolate FS_101 NADH dehydrogenase subunit 5 (ND5) gene, partial cds; NADH dehydrogenase subunit 6 (ND6) gene, complete cds; tRNA-Glu gene, complete sequence; and cytochrome b (cytb) gene, partial cds; mitochondrial	OL654361	cytochrome b
Felis catus	Felis catus MKRaS008 mitochondrial gene for cytochrome b, partial cds	LC649705	cytochrome b
Felis chaus	Felis chaus isolate Jungle Cat 5 cytochrome b (cytb) gene, partial cds; mitochondrial	MN370575	cytochrome b
Felis environmental	Felis environmental sample isolate Kw-170 cytochrome b (cytb) gene, partial cds; mitochondrial	MK510873	cytochrome b
Felis margarita	Felis margarita haplotype AH NADH dehydrogenase subunit 5 (ND5) and cytochrome b (cytb) genes, partial cds; and tRNA-Thr gene and D-loop, partial sequence; mitochondrial	MK606132	cytochrome b
Felis bieti	Felis bieti cytochrome b gene, partial cds; mitochondrial	AY773081	cytochrome b
Felis domesticus	F.domesticus mitochondrial cytochrome b gene	X82296	cytochrome b
Vulpes vulpes	Vulpes vulpes isolate LH198 haplotype FOX14 cytochrome b (CYTB) gene, partial cds; mitochondrial	MK244493	cytochrome b
Vulpes corsac	Vulpes corsac isolate SH21 cytochrome b (CYTB) gene, complete cds; mitochondrial	MT795179	cytochrome b
Vulpes zerda	Vulpes zerda isolate X161349 cytochrome b (Cytb) gene, partial cds; mitochondrial	MH854561	cytochrome b
Vulpes cana	Vulpes cana isolate B.F.Y3 cytochrome b (cytb) gene, partial cds; mitochondrial	KU378587	cytochrome b
Vulpes rueppellii	Vulpes rueppellii isolate R.F.Y6 cytochrome b (cytb) gene, partial cds; mitochondrial	KU378373	cytochrome b
Vulpes lagopus	Vulpes lagopus haplotype 5 cytochrome b (cytb) gene, partial cds; mitochondrial	KX093945	cytochrome b
Vulpes ferrilata	Vulpes ferrilata haplotype 1 cytochrome b (cytb) gene, partial cds; mitochondrial	EU872065	cytochrome b
Vulpes macrotis	Vulpes macrotis cytochrome b (cytb) gene, mitochondrial gene encoding mitochondrial protein, partial cds	AF028157	cytochrome b
Vulpes pallida	Vulpes pallida haplotype PMa2 cytochrome b (cytb) gene, partial cds; mitochondrial	KJ597964	cytochrome b
Vulpes vulpes	V.vulpes mitochondrial DNA for cytochrome b (complete sequence)	X94929	cytochrome b
Phoca largha	Phoca largha PLCBRe4 mitochondrial cytb gene for cytochrome b, partial cds	LC466149	cytochrome b
Pagophilus groenlandicus	Pagophilus groenlandicus cytochrome b gene, partial cds; mitochondrial gene for mitochondrial product	AF200491	cytochrome b
Phoca groenlandica	Phoca groenlandica cytochrome b (cytb) gene, complete cds; mitochondrial	GU174609	cytochrome b
Phoca fasciata	Phoca fasciata cytochrome b (cytb) gene, complete cds; mitochondrial	GU167294	cytochrome b
Phoca vitulina	Phoca vitulina mitochondrial cytochrome b gene, partial cds	L19127	cytochrome b
Phoca vitulina	P.vitulina mitochondrial cytochrome b gene	X82306	cytochrome b
Phoca largha	P.largha mitochondrial cytochrome b gene	X82305	cytochrome b
Phoca groenlandica	P.groenlandica mitochondrial cytochrome b gene	X82303	cytochrome b
Phoca fasciata	P.fasciata mitochondrial cytochrome b gene	X82302	cytochrome b
Manis pentadactyla	Manis pentadactyla isolate ST08 cytochrome b (cytb) gene, partial cds; mitochondrial	MW197469	cytochrome b
Felis silvestris	Felis silvestris silvestris isolate FS_101 NADH dehydrogenase subunit 5 (ND5) gene, partial cds; NADH dehydrogenase subunit 6 (ND6) gene, complete cds; tRNA-Glu gene, complete sequence; and cytochrome b (cytb) gene, partial cds; mitochondrial	OL654361	NADH dehydrogenase subunit 5
Felis catus	Felis catus isolate 4709-K NADH dehydrogenase subunit 5 (ND5) gene, partial cds; NADH dehydrogenase subunit 6 (ND6) gene, complete cds; tRNA-Glu gene, complete sequence; and cytochrome b (CYTB) gene, partial cds; mitochondrial	MN313781	NADH dehydrogenase subunit 5
Felis margarita	Felis margarita haplotype AH NADH dehydrogenase subunit 5 (ND5) and cytochrome b (cytb) genes, partial cds; and tRNA-Thr gene and D-loop, partial sequence; mitochondrial	MK606132	NADH dehydrogenase subunit 5
Felis chaus	Felis chaus isolate JCAIZ003 NADH dehydrogenase subunit 5 (ND5) gene, partial cds; mitochondrial	GU561700	NADH dehydrogenase subunit 5
Vulpes lagopus	Vulpes lagopus ATP synthase F0 subunit 6 (ATP6), ATP synthase F0 subunit 8 (ATP8), cytochrome c oxidase subunit I (COX1), cytochrome c oxidase subunit II (COX2), cytochrome c oxidase subunit III (COX3), cytochrome b (CYTB), NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 2 (ND2), NADH dehydrogenase subunit 3 (ND3), NADH dehydrogenase subunit 4 (ND4), NADH dehydrogenase subunit 4L (ND4L), and NADH dehydrogenase subunit 5 (ND5) genes, complete cds; mitochondrial	AH014073	NADH dehydrogenase subunit 5
Phoca groenlandica	Phoca groenlandica NADH dehydrogenase subunit 5 (ND5) gene, complete cds; mitochondrial gene for mitochondrial product	AY377376	NADH dehydrogenase subunit 5
Phoca fasciata	Phoca fasciata NADH dehydrogenase subunit 5 (ND5) gene, complete cds; mitochondrial	GU167331	NADH dehydrogenase subunit 5
Felis catus	Felis catus voucher N22b 12S ribosomal RNA gene, partial sequence; mitochondrial	KX786344	12S ribosomal RNA
Felis chaus	Felis chaus isolate G 12S ribosomal RNA gene, partial sequence; mitochondrial	KU963205	12S ribosomal RNA
Felis silvestris	Felis silvestris 12S ribosomal RNA gene, partial sequence; mitochondrial	KX002032	12S ribosomal RNA
Felis bieti	Felis bieti 12S ribosomal RNA gene, partial sequence; mitochondrial	AY773084	12S ribosomal RNA
Vulpes vulpes	Vulpes vulpes Vv1 mitochondrial gene for 12S ribosomal RNA, partial sequence	LC424764	12S ribosomal RNA
Vulpes lagopus	Vulpes lagopus isolate FRT12 12S ribosomal RNA gene, partial sequence; mitochondrial	KM224240	12S ribosomal RNA
Phoca fasciata	Phoca fasciata isolate 5888 12S ribosomal RNA gene, partial sequence; mitochondrial	GU174595	12S ribosomal RNA
Phoca largha	Phoca largha isolate 06spotted03 12S ribosomal RNA gene, partial sequence; mitochondrial	GU174591	12S ribosomal RNA
Manis pentadactyla	Manis pentadactyla 12S ribosomal RNA gene, partial sequence; and tRNA-Val gene, complete sequence; mitochondrial	AY012154	12S ribosomal RNA
Felis catus	Felis catus voucher Cat_KU cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	MN124254	cytochrome oxidase subunit 1
Felis nigripes	Felis nigripes voucher NZG:BWP38761 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	KX012677	cytochrome oxidase subunit 1
Felis margarita	Felis margarita voucher 198_Fe_mar cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	KF297765	cytochrome oxidase subunit 1
Vulpes vulpes	Vulpes vulpes voucher BIOUG:HBL008452 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443560	cytochrome oxidase subunit 1
Vulpes chama	Vulpes chama voucher NZG:BWP38701 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	KX012672	cytochrome oxidase subunit 1
Vulpes lagopus	Vulpes lagopus voucher HBL008485 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443554	cytochrome oxidase subunit 1
Vulpes velox	Vulpes velox voucher ROM 105399 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443557	cytochrome oxidase subunit 1
Phoca vitulina	Phoca vitulina voucher HBL008389 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443364	cytochrome oxidase subunit 1
Phoca largha	Phoca largha voucher HBL008423 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443363	cytochrome oxidase subunit 1
Phoca groenlandica	Phoca groenlandica voucher HBL008364 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial	JF443362	cytochrome oxidase subunit 1
Manis pentadactyla	Manis pentadactyla isolate KFBG_HZ0050 cytochrome oxidase subunit 1 gene, partial cds; mitochondrial	KT428152	cytochrome oxidase subunit 1
Felis catus	Felis catus growth hormone receptor (GHR) gene, partial cds	DQ205829	growth hormone receptor
Vulpes vulpes	Vulpes vulpes growth hormone receptor gene, exon 10 and partial cds	AY885401	growth hormone receptor
Vulpes macrotis	Vulpes macrotis growth hormone receptor gene, exon 10 and partial cds	AY885400	growth hormone receptor
Vulpes corsac	Vulpes corsac growth hormone receptor gene, exon 10 and partial cds	AY885399	growth hormone receptor
Vulpes zerda	Vulpes zerda growth hormone receptor gene, exon 10 and partial cds	AY885393	growth hormone receptor
Alopex lagopus	Alopex lagopus growth hormone receptor gene, exon 10 and partial cds	AY885379	growth hormone receptor
Vulpes velox	Vulpes velox growth hormone receptor (GHR) gene, partial cds	DQ205838	growth hormone receptor
Vulpes lagopus	Vulpes lagopus growth hormone receptor (GHR) gene, partial cds	DQ205837	growth hormone receptor
Phoca vitulina	Phoca vitulina growth hormone receptor (GHR) gene, partial cds	GU931127	growth hormone receptor
Phoca largha	Phoca largha growth hormone receptor (GHR) gene, partial cds	DQ205827	growth hormone receptor
Phoca groenlandica	Phoca groenlandica growth hormone receptor (GHR) gene, partial cds	DQ205825	growth hormone receptor
Manis pentadactyla	Manis pentadactyla growth hormone receptor (GHR) gene, exon 10 and partial cds	EU448992	growth hormone receptor
Felis catus	Felis catus interphotoreceptor retinoid binding protein gene, exon 1	Z11811	interphotoreceptor retinoid-binding protein
Vulpes velox	Vulpes velox interphotoreceptor retinoid binding protein gene, partial cds	AF179293	interphotoreceptor retinoid-binding protein
Manis pentadactyla	Manis pentadactyla interphotoreceptor retinoid binding protein (IRBP) gene, exon 1 and partial cds	JN414784	interphotoreceptor retinoid-binding protein
Felis silvestris	Felis silvestris haplotype W23 tRNA-Pro gene and control region, partial sequence; mitochondrial	MF353436	tRNA-Pro
Felis catus	Felis catus mitochondrial tRNA-Pro gene and control region, partial sequence	AF348642	tRNA-Pro
Vulpes vulpes	Vulpes vulpes isolate VvUAE056 tRNA-Thr gene, partial sequence; tRNA-Pro gene, complete sequence; and D-loop, partial sequence; mitochondrial	MT955893	tRNA-Pro
Vulpes rueppellii	Vulpes rueppellii isolate VrKSAA0005 tRNA-Thr gene, partial sequence; tRNA-Pro gene, complete sequence; and D-loop, partial sequence; mitochondrial	MT955814	tRNA-Pro
Vulpes lagopus	Vulpes lagopus haplotype H11 tRNA-Pro gene and D-loop, partial sequence; mitochondrial	KX093931	tRNA-Pro
Vulpes macrotis	Vulpes macrotis haplotype Vmac1 tRNA-Pro gene and D-loop, partial sequence; mitochondrial	KJ846673	tRNA-Pro
Vulpes zerda	Vulpes zerda haplotype Vzer1 tRNA-Pro gene and D-loop, partial sequence; mitochondrial	KJ846672	tRNA-Pro
Vulpes ferrilata	Vulpes ferrilata haplotype 4 D-loop, partial sequence; tRNA-Pro gene, complete sequence; and tRNA-Thr gene, partial sequence; mitochondrial	JF520840	tRNA-Pro
Phoca largha	Phoca largha isolate pl27 tRNA-Thr gene, partial sequence; tRNA-Pro gene, complete sequence; and D-loop, partial sequence; mitochondrial	OM967017	tRNA-Pro
Phoca vitulina	Phoca vitulina isolate N tRNA-Thr (trnT) gene, partial sequence; tRNA-Pro (trnP) gene, complete sequence; and D-loop, partial sequence; mitochondrial	HQ702987	tRNA-Pro
Manis pentadactyla	Manis pentadactyla pentadactyla isolate MPP5 tRNA-Pro gene, partial sequence; D-loop, complete sequence; and tRNA-Phe gene, partial sequence; mitochondrial	GQ232081	tRNA-Pro

Now, since we’re going to retrieve sequences from genbank using an existing preliminary accession numbers table, we will use the sq.retrieve.indirect() function in phruta. I’m going to spend some time in here to explain the differences between the two versions of sq.retrieve.* in phruta. The one that we’re using in this tutorial, sq.retrieve.indirect(), retrieves sequences “indirectly” because it follows the initial step of generating a table summarizing accession numbers (see the acc.table.retrieve() function above). I present the information in this vignette using sq.retrieve.indirect() instead of sq.retrieve.direct() because the first function is way more flexible and allows for correcting issues prior to download any sequence. For instance, you can add new sequences, species, populations to the resulting data.frame from acc.table.retrieve(). Additionally, you could even manually assemble your own dataset of accession numbers to be retrieved using sq.retrieve.indirect(). Instead, sq.retrieve.direct() does its best to directly (i.e. without potential input from the user) retrieve sequences for a target set of taxa and set of gene regions. In short, you should be able to catch errors using sq.retrieve.indirect() but mistakes will be harder to spot and fix if you’re using sq.retrieve.direct(). Note that the functionality of sq.retrieve.direct() is outlined in the “Using phruta with defined target genes” vignette.

We still need to retrieve all the sequences from the accessions table that was generated avobe using acc.table. The sq.retrieve.indirect() function will write all the resulting fasta files into a newly created folder 0.Sequences located in our working directory (please check the download.sqs = TRUE argument).

sq.retrieve.indirect(acc.table, download.sqs = TRUE)

Next, we’re going to make sure that we include only sequences that are reliable and from species that we are actually interested in analyzing. For this, we will be using the sq.curate() function. We need to provide a list of taxonomic names to filter out incorrect sequences (filterTaxonomicCriteria argument). For simplicity, our criteria can be the genera that we’re interested in analyzing. Note that the outgroup’s name should also be included in the list. If the taxonomic information for a sequence retrieved from genbank does not match with any of these strings, this species will be dropped. You will have to specify whether sampling is for animals or plants (kingdom argument). Finally, you might have already noticed that the same gene regions can have different names. For instance, sometimes our searches retrieve both “cytochrome oxidase subunit 1” and “cytochrome c oxidase subunit I” as widely sampled genes for the target species. In that case, we can combine the sequences in these two files into a single file name COI. To merge gene files, you will have to provide a named list to the mergeGeneFiles argument of the sq.curate function. This named list (tb.merged below) will have a length that corresponds to the number of final files that should be constructed.

tb.merged <- list('COI' = c("cytochrome oxidase subunit 1", "cytochrome c oxidase subunit I"))
sq.curate(filterTaxonomicCriteria = 'Felis|Vulpes|Phoca|Manis',
          mergeGeneFiles = tb.merged,
          kingdom = 'animals', 
          folder = '0.Sequences',
          removeOutliers = FALSE)

Running the line of code above will create the a folder 1.CuratedSequences containing (1) the curated sequences with original names, (2) the curated sequences with species-level names (renamed_* prefix), (3) a table of accession numbers (0.AccessionTable.csv), and (4) a summary of the taxonomic information for all the species sampled in the files (1.Taxonomy.csv). We’ll use the renamed_* and 1.Taxonomy.csv files in the next steps. Let’s take a look at the sampling per gene region in the 0.AccessionTable.csv table.

4. Preliminary accession number table
OriginalNames	AccN	Species	file	OldSpecies
KX786344 Felis catus	KX786344	Felis_catus	12S ribosomal RNA.fasta	Felis_catus
KU963205 Felis chaus	KU963205	Felis_chaus	12S ribosomal RNA.fasta	Felis_chaus
KX002032 Felis silvestris	KX002032	Felis_silvestris	12S ribosomal RNA.fasta	Felis_silvestris
AY773084 Felis bieti	AY773084	Felis_bieti	12S ribosomal RNA.fasta	Felis_bieti
LC424764 Vulpes vulpes	LC424764	Vulpes_vulpes	12S ribosomal RNA.fasta	Vulpes_vulpes
KM224240 Vulpes lagopus	KM224240	Vulpes_lagopus	12S ribosomal RNA.fasta	Vulpes_lagopus
GU174595 Phoca fasciata	GU174595	Histriophoca_fasciata	12S ribosomal RNA.fasta	Phoca_fasciata
GU174591 Phoca largha	GU174591	Phoca_largha	12S ribosomal RNA.fasta	Phoca_largha
AY012154 Manis pentadactyla	AY012154	Manis_pentadactyla	12S ribosomal RNA.fasta	Manis_pentadactyla
OL654361 Felis silvestris	OL654361	Felis_silvestris	cytochrome b.fasta	Felis_silvestris
LC649705 Felis catus	LC649705	Felis_catus	cytochrome b.fasta	Felis_catus
MN370575 Felis chaus	MN370575	Felis_chaus	cytochrome b.fasta	Felis_chaus
MK606132 Felis margarita	MK606132	Felis_margarita	cytochrome b.fasta	Felis_margarita
AY773081 Felis bieti	AY773081	Felis_bieti	cytochrome b.fasta	Felis_bieti
MK244493 Vulpes vulpes	MK244493	Vulpes_vulpes	cytochrome b.fasta	Vulpes_vulpes
MT795179 Vulpes corsac	MT795179	Vulpes_corsac	cytochrome b.fasta	Vulpes_corsac
MH854561 Vulpes zerda	MH854561	Vulpes_zerda	cytochrome b.fasta	Vulpes_zerda
KU378587 Vulpes cana	KU378587	Vulpes_cana	cytochrome b.fasta	Vulpes_cana
KU378373 Vulpes rueppellii	KU378373	Vulpes_rueppellii	cytochrome b.fasta	Vulpes_rueppellii
KX093945 Vulpes lagopus	KX093945	Vulpes_lagopus	cytochrome b.fasta	Vulpes_lagopus
EU872065 Vulpes ferrilata	EU872065	Vulpes_ferrilata	cytochrome b.fasta	Vulpes_ferrilata
AF028157 Vulpes macrotis	AF028157	Vulpes_macrotis	cytochrome b.fasta	Vulpes_macrotis
KJ597964 Vulpes pallida	KJ597964	Vulpes_pallida	cytochrome b.fasta	Vulpes_pallida
LC466149 Phoca largha	LC466149	Phoca_largha	cytochrome b.fasta	Phoca_largha
GU174609 Phoca groenlandica	GU174609	Pagophilus_groenlandicus	cytochrome b.fasta	Phoca_groenlandica
GU167294 Phoca fasciata	GU167294	Histriophoca_fasciata	cytochrome b.fasta	Phoca_fasciata
L19127 Phoca vitulina	L19127	Phoca_vitulina	cytochrome b.fasta	Phoca_vitulina
MW197469 Manis pentadactyla	MW197469	Manis_pentadactyla	cytochrome b.fasta	Manis_pentadactyla
MN124254 Felis catus	MN124254	Felis_catus	cytochrome oxidase subunit 1.fasta	Felis_catus
KX012677 Felis nigripes	KX012677	Felis_nigripes	cytochrome oxidase subunit 1.fasta	Felis_nigripes
KF297765 Felis margarita	KF297765	Felis_margarita	cytochrome oxidase subunit 1.fasta	Felis_margarita
JF443560 Vulpes vulpes	JF443560	Vulpes_vulpes	cytochrome oxidase subunit 1.fasta	Vulpes_vulpes
KX012672 Vulpes chama	KX012672	Vulpes_chama	cytochrome oxidase subunit 1.fasta	Vulpes_chama
JF443554 Vulpes lagopus	JF443554	Vulpes_lagopus	cytochrome oxidase subunit 1.fasta	Vulpes_lagopus
JF443557 Vulpes velox	JF443557	Vulpes_velox	cytochrome oxidase subunit 1.fasta	Vulpes_velox
JF443364 Phoca vitulina	JF443364	Phoca_vitulina	cytochrome oxidase subunit 1.fasta	Phoca_vitulina
JF443363 Phoca largha	JF443363	Phoca_largha	cytochrome oxidase subunit 1.fasta	Phoca_largha
JF443362 Phoca groenlandica	JF443362	Pagophilus_groenlandicus	cytochrome oxidase subunit 1.fasta	Phoca_groenlandica
KT428152 Manis pentadactyla	KT428152	Manis_pentadactyla	cytochrome oxidase subunit 1.fasta	Manis_pentadactyla
DQ205829 Felis catus	DQ205829	Felis_catus	growth hormone receptor.fasta	Felis_catus
AY885401 Vulpes vulpes	AY885401	Vulpes_vulpes	growth hormone receptor.fasta	Vulpes_vulpes
AY885400 Vulpes macrotis	AY885400	Vulpes_macrotis	growth hormone receptor.fasta	Vulpes_macrotis
AY885399 Vulpes corsac	AY885399	Vulpes_corsac	growth hormone receptor.fasta	Vulpes_corsac
AY885393 Vulpes zerda	AY885393	Vulpes_zerda	growth hormone receptor.fasta	Vulpes_zerda
AY885379 Alopex lagopus	AY885379	Vulpes_lagopus	growth hormone receptor.fasta	Alopex_lagopus
DQ205838 Vulpes velox	DQ205838	Vulpes_velox	growth hormone receptor.fasta	Vulpes_velox
GU931127 Phoca vitulina	GU931127	Phoca_vitulina	growth hormone receptor.fasta	Phoca_vitulina
DQ205827 Phoca largha	DQ205827	Phoca_largha	growth hormone receptor.fasta	Phoca_largha
DQ205825 Phoca groenlandica	DQ205825	Pagophilus_groenlandicus	growth hormone receptor.fasta	Phoca_groenlandica
EU448992 Manis pentadactyla	EU448992	Manis_pentadactyla	growth hormone receptor.fasta	Manis_pentadactyla
OL654361 Felis silvestris	OL654361	Felis_silvestris	NADH dehydrogenase subunit 5.fasta	Felis_silvestris
MN313781 Felis catus	MN313781	Felis_catus	NADH dehydrogenase subunit 5.fasta	Felis_catus
MK606132 Felis margarita	MK606132	Felis_margarita	NADH dehydrogenase subunit 5.fasta	Felis_margarita
GU561700 Felis chaus	GU561700	Felis_chaus	NADH dehydrogenase subunit 5.fasta	Felis_chaus
AH014073 Vulpes lagopus	AH014073	Vulpes_lagopus	NADH dehydrogenase subunit 5.fasta	Vulpes_lagopus
AY377376 Phoca groenlandica	AY377376	Pagophilus_groenlandicus	NADH dehydrogenase subunit 5.fasta	Phoca_groenlandica
GU167331 Phoca fasciata	GU167331	Histriophoca_fasciata	NADH dehydrogenase subunit 5.fasta	Phoca_fasciata
MF353436 Felis silvestris	MF353436	Felis_silvestris	tRNA-Pro.fasta	Felis_silvestris
AF348642 Felis catus	AF348642	Felis_catus	tRNA-Pro.fasta	Felis_catus
MT955893 Vulpes vulpes	MT955893	Vulpes_vulpes	tRNA-Pro.fasta	Vulpes_vulpes
MT955814 Vulpes rueppellii	MT955814	Vulpes_rueppellii	tRNA-Pro.fasta	Vulpes_rueppellii
KX093931 Vulpes lagopus	KX093931	Vulpes_lagopus	tRNA-Pro.fasta	Vulpes_lagopus
KJ846673 Vulpes macrotis	KJ846673	Vulpes_macrotis	tRNA-Pro.fasta	Vulpes_macrotis
KJ846672 Vulpes zerda	KJ846672	Vulpes_zerda	tRNA-Pro.fasta	Vulpes_zerda
JF520840 Vulpes ferrilata	JF520840	Vulpes_ferrilata	tRNA-Pro.fasta	Vulpes_ferrilata
OM967017 Phoca largha	OM967017	Phoca_largha	tRNA-Pro.fasta	Phoca_largha
HQ702987 Phoca vitulina	HQ702987	Phoca_vitulina	tRNA-Pro.fasta	Phoca_vitulina
GQ232081 Manis pentadactyla	GQ232081	Manis_pentadactyla	tRNA-Pro.fasta	Manis_pentadactyla

We’ll now align the sequences that we just curated. For this, we just use sq.aln() with default parameters. We need to indicate that we’re interested in aligning only the "renamed" fasta files in our 1.CuratedSequences folder.

sq.aln(folder = '1.CuratedSequences', FilePatterns = "renamed")

The resulting multiple sequence alignments will be saved to the 2.Alignments folder. In that new folder, we will have two types of files: (1) raw alignments (same file names as in 1.CuratedSequences) and (2) alignments with ambiguous sites removed (Masked_* prefix). Masked alignments are only created if the mask argument in sq.aln is set to TRUE. In that case, one additional .csv file is created for each of the alignments (0.Masked.Information_*). Each of these datasets list the number of sites in the masked alignment that (1) are not gaps (NonGaps column), (2) if the sequence was removed due to the elevated number of gaps (removedPerGaps; controlled using the threshold argument in sq.aln), or (3) if it was removed directly in the masking step (removedMasking). Note that, for some gene regions, making can fail. In that case, only the original alignment file is saved to the 2.Alignments folder.

Note that we could use these resulting alignments directly to infer our phylogenies. We cover these steps within phruta in another vignette: “Phylogenetics with the phruta R package”. For now, let’s wrap up and plot one of our (cool) alignments. Let’s first check the raw alignments!

A figure showing raw alignments

Now, the masked alignments…!!

A figure showing curated alignments

And we’re done for now!! Thanks for following this tutorial…:)

In total, this vignette took 11 minutes to render in my local machine. You can now try to run phruta using your favorite groups organisms! Don’t forget to check the other tutorials and get in touch if you find any issues…Buena suerte!

Table of Contents

To export or not export from phruta

From taxonomic names to sequence alignments, exporting data

To export or not export from `phruta`