Phylogenomic Analysis Pipeline for Herbarium Specimens
What is PhyloHerb: PhyloHerb is a wrapper program to process genome skimming data collected from plant materials. The outcomes include the plastid genome (plastome) assemblies, mitochondrial genome assemblies, nuclear ribosomal DNAs (NTS+ETS+18S+ITS1+5.8S+ITS2+28S), alignments of gene and intergenic regions, and a species tree. It is designed to be a high throughput program dealing with lower quality data. Examples include low-coverage (5x cpDNA) plastome phylogeny, recycling plastid genes from target enrichment data, retrieving low-copy nuclear genes from medium coverage (5x nucDNA) genome skimming.
License: GNU General Public License
Citation:
- Cai, Liming, Hongrui Zhang, and Charles C. Davis. 2022. PhyloHerb: A high‐throughput phylogenomic pipeline for processing genome‐skimming data. Applications in Plant Sciences 10(3): 1–9. https://doi.org/10.1002/aps3.11475
PhyloHerb relies on the following dependancies:
-
Jin, Jian-Jun, Wen-Bin Yu, Jun-Bo Yang, Yu Song, Claude W. Depamphilis, Ting-Shuang Yi, and De-Zhu Li. "GetOrganelle: a fast and versatile toolkit for accurate de novo assembly of organelle genomes." Genome biology 21, no. 1 (2020): 1-31.
-
Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y., McGinnis, S., & Madden, T. L. (2008). NCBI BLAST: a better web interface. Nucleic acids research, 36(suppl_2), W5-W9.
See you in Evolution June 24-28, 2022: Liming will be presenting a in-person talk and poster on PhyloHerb at Evolution. See you in Cleveland! The poster is deposited online here.
New workshop July 24, 2022: We will hold an in-person workshop (with remote options) at Botany 2022 in Anchorage. See the BSA website for abstract. Workshop materials can be found here.
New features released: Since PhyloHerb v1.1+, you can pull low-copy nuclear genes (e.g., Angiosperm 353) from your genome skimming data. All you need is the reference sequences and raw reads. PhyloHerb will output fasta files that are ready for alignment. Check the low-copy nuclear gene tutorial below.
New publication: Initial PhyloHerb release accepted at APPS and we are selected as the cover! Stay tuned for the early view! 🎉
I. Prerequisites and installation
III. Complete tutorial for analyzing genome skimming data for phylogenetic analysis
IV. Retrieve low-copy nuclear genes
V. General guidelines for genome skimming data collection
To process large datasets (>20 sp), high performance cluster is recommended. Mac and PC may suffer from insufficient memory during the assembly, alignment, or phylogenetic reconstruction. If you have difficulties installing PhyloHerb, please contact Liming Cai ([email protected]) or open an Issue.
IMPORTANT: PhyloHerb is currently only compatible with Python 3.
Installation via conda
Create a conda environment under Python 3 and activate the environment
#install blast, biopython, bowtie2, spades, samtools, pandas, and ete3
conda install -c bioconda blast
conda install -c conda-forge biopython
conda install -c etetoolkit ete3
conda install -c bioconda bowtie2
conda install -c bioconda spades
conda install -c bioconda samtools
#please make sure panads is <2.0
conda install pandas=1.5.3
To install PhyloHerb, simply download it use git:
git clone https://github.com/lmcai/PhyloHerb.git
Or from the source package:
#for v1.1.2
wget https://github.com/lmcai/PhyloHerb/archive/refs/tags/phyloherb_v1.1.2.tar.gz
tar xzvf phyloherb_v1.1.2.tar.gz
To update your local version for any future releases, cd into the PhyloHerb directory then type
git fetch --prune origin
git reset --hard origin
git clean -f -d
Alternative: Install dependencies separately from source
You could also want to install these dependencies from source. If you are using computer clusters, some dependencies might also be installed and can be called via module load. Make sure all dependencies are callable in your current environment. A list of PhyloHerb dependencies:
Then download and decompress the PhyloHerb python package.
-
Assembler: GetOrganelle v1.7.0+. Most easily installed via
conda. All of the dependancies will be installed automatically. -
Assembly viewer: Bandage
-
Optional assembly viewer: Geneious (the complementary version is sufficient)
-
Aligner:
Pasta for highly variable regions such as the ITS sequences
MAFFT for less variable regions or long alignments (>5 kb) that pasta may not be able to handle when the number of species is high (>500 sp)
-
Manual assembly examination: Geneious (the licensed version are required)
Core functions of PhyloHerb
phyloherb.py [-h] -m mode [-i dir] [-o dir] [-suffix string] [-sp file]
[-g file] [-l integer] [-evalue float] [-n integer]
[-ref file] [-mito] [-rdna] [-nuc] [-r1 read file]
[-r2 read file] [-rs read file] [-prefix string] [-t file]
[-missing float 0-1] [-f mode] [-gene_def file] [-b file]
[-s file]
-h, --help show this help message and exit
-m mode execution mode, choose one of the following: qc, ortho,
conc, order, getseq, assemb, submission
-i dir input directory
-o dir output directory
-suffix string [qc, ortho, conc mode] suffix of input files
-sp file [ortho mode] a file containing a list of species
-g file [ortho and conc mode] a file containing a list of loci
-l integer [ortho mode] minimum length of blast hits
-evalue float [ortho mode] evalue threshold for BLAST
-n integer [ortho, assemb mode] number of threads
-ref file [ortho mode] custom reference sequences
-mito [ortho mode] extract mitochondrial genes using build-in
references
-rdna [ortho mode] extract nuclear ribosomal regions using
build-in references
-nuc [ortho mode] extract low-copy nuclear loci
-r1 reads file [assemb mode] forward reads
-r2 reads file [assemb mode] reverse reads
-rs reads file [assemb mode] single-end or unpaired reads, separate
mulitple file by ','
-prefix string [assemb mode] assembly output prefix
-t file [order mode] newick tree file to order alignments based
on phylogeny
-missing float 0-1 [order mode] maximum proportion of missing data allowed
for each species
-f mode [getseq mode] how to extract loci, choose one of the
following: gene, genetic_block, intergenic
-gene_def file [getseq mode] a gene delimitation file that defines
genetic blocks
-b file [submission mode] path to the bash file
-s file [submission mode] path to the taxon sampling sheet
1. Ortholog gene extraction (cpDNA, rDNA, mtDNA) using built-in database
A. If you have your organellar or rDNA assemblies and want to extract genes using our curated database:
Input: Place all fasta formated assemblies in one folder. Make sure they have consistent suffix (e.g., .fas, .fasta). To extract genes, use the following command:
#For plastid
python phyloherb.py -m ortho -i <input directory> -o <output directory> -suffix <suffix>
#For rDNA
python phyloherb.py -m ortho -i <input directory> -o <output directory> -suffix <suffix> -rdna
#For mitochondrion
python phyloherb.py -m ortho -i <input directory> -o <output directory> -suffix <suffix> -mito
List of genes and species included in our built-in database can be found here.
Output: In the output folder, you can find fasta sequences named after genes. The header within each fasta is consistent with the species names (file names of the input assemblies).
The 30-second gif image below shows the time and CPU to extract all plastid genes for 100 species —— it only takes half a minute.
B. If you need to assemble organelle genomes and rDNA regions, see Section III.1 Assembly below.
2. Ortholog gene extraction for low-copy nuclear genes with custom references
Input: One fasta file of reference genes, formatted following the custom reference guidance. Raw reads of target species, pair-end or single-end. This function is similar to a quick-and-dirty version of HybPiper and involves two-steps: spades assembly and ortholog extraction.
To assemble target regions, use the following command for each species:
python phyloherb.py -m assemb -r1 <Forward.fq> -r2 <Reverse.fq> -rs <Single1.fq,Single2.fq> -ref
<reference fasta> -prefix <species ID/name> -n <threads>
Once the assemblies are completed for all species, generate ortholog fasta files:
python phyloherb.py -m ortho -i <input directory> -o <output directory> -suffix <suffix> -ref reference.fasta -nuc
The input directory should be the parent directory containing all spades output folders.
Output: In the output folder, you will find fasta sequences named after genes. The header within each fasta is consistent with the species IDs/names in the assembly step (file names of the spades output folder).
3. Alignment and concatenation
For small dataset in conserved regions, you can use MAFFT for align. For large dataset across distantly related species, we recommend PASTA. An example bash file to run MAFFT and PASTA can be found in mafft_pasta.sh. To concatenate sequences, place all fasta alignments in one folder and use the following command:
python phyloherb.py -m conc -i < input directory> -o <output prefix> -suffix <alignment suffix>
Output: *.conc.fas and *.conc.nex are concatenated sequences in fasta and nexus formats, respectively. *.partition is the gene partition file that can be used for PartitionFinder.
4. Reorder alignments based on phylogeny to assist manual curation
Reorder sequences based on phylogeny can help distinguish analytical errors versus shared mutations.
Input: One species tree in newick format; multiple FASTA alignments in one folder.
python phyloherb.py -m order -t <tree> -i <input dir> -o <output dir> -suffix <alignment suffix>
Output: *.ordered.fas is the reordered alignment for each gene. *.pasta_ref.tre is the pruned species tree that can be used as the reference tree in the second PASTA alignment.
We will use GetOrganelle to assemble the plastome, mitochondrial genome, and ribosomal regions. It requires minimal tweak for various types of data. I highly recommend installing it using conda so that all its dependencies are in your environment.
Illumina FASTQ reads for each species, single-ended or pair-ended, zipped or unzipped. We recommend trimming the adapters, but not filtering the reads based on quality.
After loading GetOrganelle to your environment, the basic commands for running assembly with pair end data is as follows:
#To assemble plant plastome
get_organelle_from_reads.py -1 <forward.fq> -2 <reverse.fq> -o <plastome_output> -R 15 -k 21,45,65,85,95,105 -F embplant_pt
#To assemble plant nuclear ribosomal RNA
get_organelle_from_reads.py -1 <forward.fq> -2 <reverse.fq> -o <nr_output> -R 10 -k 35,85,105,115 -F embplant_nr
#To assemble plant mitochondria:
get_organelle_from_reads.py -1 <forward.fq> -2 <reverse.fq> -o <mito_output> -R 50 -k 21,45,65,85,105 -P 1000000 -F embplant_mt
If you want to use your own reference sequences for assembly, you can provide the seed fasta file by adding -ref <reference.fas>.
If you are working with a high performance computing cluster with slurm workload manager, you can submit individual assembly tasks to the cluster and run them simultaneously. An example bash file is provided in /phyloherbLib/getorg.sh. You can submit your job by typing
sbatch getorg.sh <forward.fq> <backward.fq> <output prefix>
For more details using this bash file, see the tutorial.
IMPORTANT: Make sure you load the correct environment and provide absolute path to the input data if they are not in the current directory by modifying relavant variables in getorg.sh. Instructions for single-end data can also be found in getorg.sh.
The key output files from Getorganelle include
*.path_sequence.fasta: the assembly in fasta format
extended_K*.assembly_graph.fastg.extend_embplant_pt-embplant_mt.fastg: a simplified assembly graph
extended_K*.assembly_graph.fastg.extend_embplant_pt-embplant_mt.csv: a tab-format contig label file for bandage visualization
get_org.log.txt: the log file
Bandage is a program for visualising de novo assembly graphs. The assembly graph files *.fastg and *.gfa generated from GetOrganelle be visualized in Bandage and exported into sequences.
The *.path_sequence.fasta files do not always navigate the right paths for organelle genomes, especially the ones with complicated structures. The authors of GetOrganelle put together a nice video introducing how to generate complete (if possible) and accurate sequences from Bandage with different examples.
After the assemblies are completed, you can summarize the results using the qc function of phyloherb.
python phyloherb.py -m qc -i <parent dir containing Getorganelle output folders> -o <output dir>
Output:
*.assembly.fas is the fasta files of all assemblies. assembly_sum.tsv is the summary spreadsheet with the following information:
sp_prefix Total_reads Reads_in_target_region Average_base_coverage Length GC% Number_scaffolds Circularized
Important: If your input directory contains fasta assemblies alone, only the following information will be included:
sp_prefix Length GC% Number_scaffolds
Annotation is not necessary if you are interested in phylogeny alone, but if you want to submit your circularized assemblies to GenBank or extract intergenic regions from your spcecies, it is a must.
The most convenient tool I have used is the web-based tool GeSeq. I have concatenated 100 plastomes into a single fasta file and annotated them all at once on GeSeq. But if you are annotating hundreds of plastomes, the command-line based tool PGA might be a better option.
Phyloherb will identify the best-matching region of each locus in the assemblly using BLAST. We provide a build-in database of plastid genes from 355 seed plant families. You can also supply your own reference in a fasta file following the instructions below. The list of reference species is here. The list of the genes in the database is here.
You can obtain gene sequences from GenBank or GeSeq annotations. All reference sequences should be in a single fasta file gene_ref.fas. It is recommended that for diverse groups, you use sequences from more than one species for each gene.
In gene_ref.fas, the headers should begin by the gene name then followed by an underscore and a species name (which will be ignored):
>gene1_sp1
atcg...
>gene1_sp2
atcg...
>gene2_sp1
atcg...
>gene2_sp2
IMPORTANT: If you have annotated plastid assemblies (e.g., from GeqSeq) in genbank format, you can use the getseq function of PhyloHerb to obtain gene and intergenic regions.
We can extract the target gene regions using the ortho function of phyloherb. This function will conduct BLAST search in the assembly, extract the best matching regions, and output the fasta files to a directory.
python phyloherb.py -m ortho -i <input dir with fasta files> -o <output dir>
Output: *.fas files named after genes. Within each fasta file, the headers will be the species prefixes.
You can choose to use a subset of genes and species by supplying a -g gene_subset.txt and -sp species_subset.txt. Example files can be found in gene_subset.txt and species_subset.txt. You can also set a minimum length limit for gene region extraction via -l <lower limit>. Blast hits shorter than this will not be use.
python phyloherb.py -m ortho -i <input dir with fasta files> -o <output dir> -g <gene list> -sp <species list> -l <length limit> -ref <ref seq>
PhyloHerb can extract the coding regions of the rDNA repeat (18S+ITS1+5.8S+ITS2+28S), but not NTS or ETS. The output contains five fasta files 18S.fas, ITS1.fas, 5.8S.fas, ITS2.fas, and 28S.fas in the output directory. To get rDNA sequences, add the -rdna flag under the ortho mode.
python phyloherb.py -m ortho -i <input dir> -o <output dir> -rdna
Below is an illustration of the structure and sequence conservation of the nuclear ribosomal region.
Mitochondrial genes are highly conserved in plants and are not phylogenetically informative. If you want to use our build-in mitochondrial gene sequence database, invoke the -mito flag under the ortho mode.
A list of the reference mitochondrial genes can be found here. The reference sequences themselves can be found here.
python phyloherb.py -m ortho -i <input dir> -o <output dir> -mito
Intergenic regions are mostly useful for phylogenetic research among closely related species. In addition, combining short genes and intergenic regions into longer genetic blocks can reduce false positive BLAST hit. Make sure there are no structural variations in the target region.
PhyloHerb offers three modes for genetic region extraction from Genbank annotations. The results can be used as reference sequences for ortholog extraction using the ortho function of PhyloHerb.
Let's assume that there are seven genes G1-7 on a scaffold.
- The
-f genemode will extract all annotated gene features from all genbank files in the input directory
python phyloherb.py -m getseq -f gene -i <input directory> -suffix <genbank suffix> -o <output directory>
Both the -f genetic_block and -f intergenic mode will take a genetic block definition file supplied by -gene_def, and extract corresponding regions. This tab-delimited file have three columns: genetic region name, start gene, and end gene. An example can be found here.
name start end
LOC1 G1 G2
LOC2 G3 G7
...
- The
-f genetic_blockmode will extract the coding regions of the start and end genes as well as everything in between. It is good for combining multiple short genes.
python phyloherb.py -m getseq -f genetic_block -i <input directory> -suffix <genbank suffix> -o <output directory> -gene_def <gene definition file>
- Finally, the
-f intergenicmode generates similar outcomes, but does not include the genes on both ends. It is good for extracting long intergenic regions.
python phyloherb.py -m getseq -f intergenic -i <input directory> -suffix <genbank suffix> -o <output directory> -gene_def <gene definition file>
I like to use the --adjustdirection function from MAFFT to correct reverse complimentary sequences. Then I will use pasta to more accurately align high variable sequences such as the intergenic regions and the ITS regions. pasta first generates a guidance tree, then align among closely-related species, finally merge the alignments to produce the output.
This is a potentially time consuming step so I recommend running it on the cluster using the example batch file mafft_pasta.sh.
Copy mafft_pasta.sh to the same directory where the gene sequences are located. Modify the file to include appropriate environmental parameters. Then the batch job can be submitted to the cluster by typing
sbatch mafft_pasta.sh <gene_1>
sbatch mafft_pasta.sh <gene_2>
At this point, it is recommended that you take a initial look at your alignments. Be prepared to complete the "alignment-manual check-phylogeny" cycle for at least two rounds to get publication quality data.
The purpose of the initial check is to remove obvious low-quality sequences. Do not conduct any site-based filtering yet! For example, the two sequences highlighted in red below contain too many SNPs (marked in black). They should be removed.
Geneious provides a nice interface to work with alignments. You can view alignment statistics, edit sequences, and concatenate alignments. Alternative automatic tools include trimAL and Seaview.
Many tools are available for concatenating alignments. I recommend the conc function of phyloherb or Geneious. I have applied both tools to dataset with 1000 sp x 100 genes. The conc function of phyloherb will also output a gene delineation file required by PartitionFinder.
To use the conc function of phyloherb, use the following command:
python phyloherb.py -m conc -i <directory containing alignments> -o <output prefix> -suffix <suffix>
This command will concatenate all of the fasta sequences in the input directory with the specified suffix. If you only want to use a subset of the genes or want the genes to appear in a specific order, you can supply a gene order file by adding -g gene_subset.txt.
For an initial quick and dirty analysis, I recommend ExaML with unpartitioned alignment. IQTREE or RAxML generates more accurate estimations of the phylogeny and substitution paramters, but may not accomodate thousands of species with millions of sites. The commands I use for ExaML analysis is as follows.
#generate a parsimony tree with RAxML
raxmlHPC-SSE3 -y -m GTRGAMMA -s DNA_algnment.fas -n test -p 3256179
parse-examl -s DNA_aln.phy -n test -m GTRGAMMA
examl-AVX -S -s test.binary -m GAMMA -n testML -t RAxML_parsimonyTree.test
It can be a quite satisfying experience when you browse through a well-curated alignment. To get us there, we need to conduct a second round of alignment curation and remove spurious regions arising from assembly errors or false positive BLAST hits.
First, using a reference ExaML species tree (newick format), we will order the sequences based on their phylogenetic affinity. This will facilitate manual curation of the alignments in Geneious because you can see shared mutations in closely related species.
The order function of phyloherb takes a reference tree and reorders all alignments in the input directory based on the phylogeny. If you want to additionally filter sequences based on missing data, using the optional -missing flag. A float number from 0 to 1 is required for -missing to indicate the maximum proportion of ambiguous sites allowed for each sequence.
python phyloherb.py -m order -t <reference.tre> -i <directory containing alignments> -o <output directory> -suffix <suffix> [optional] -missing <float 0 to 1>
This will generate an ordered alignment *.ordered.fas and a companion tree file *.pasta_ref.tre for each gene. You will need this tree for the second round of pasta alignment after manual curation.
Now let's load the ordered alignments to Geneious for some fine tuning. This time we will delete blocks of problematic sequences. They usually appears as a cluster of SNPs highlighted in red below. These SNPs are not conserved in their close relatives, so they are phylogenetically uninformative autapomorphies. Regardless of the causes, we can safely delete them.
After a second manual check, your alignments is ready for re-alignment in pasta. This time we will use a reference tree *.pasta_ref.tre to guide the alignment for each gene.
run_pasta.py -i <input sequence> -a -t <reference tree> -o <output directory>
To submit batch job to the cluster, you can modify this batch file. Make sure you have loaded the correct environment.
When the alignment is done, you can use them to produce your final species tree.
PhyloHerb uses an mapping-assembly-scaffold approach to generate sequences of low-copy nuclear loci. It can be interpreted as a quick-and-dirty version of HybPiper. It has the advantage of rapidly pulling out target regions with low computational cost. The references sequences can be genes or CDS (e.g., Angiosperm 353 or 1kp data), but it has to be DNA seqs.
- PhyloHerb will remove the flanking 'splash zone' that is not included in the reference region.
- PhyloHerb will output no more than ONE sequence per gene per species. The combination of paralogs and missing data can generate chimeric assembly (see the illustration above). It is thus advised to use stringent BLAST evalue threshold (default 1e-40).
- Given the low coverage and non-enrichment nature of genome skimming, missing data is expected.
It is recommended to use more than one species per gene. All reference sequences should be in a single fasta file. In this files, the headers should begin by the gene name then followed by an underscore and a species name (which will be ignored):
>gene1_sp1
atcg...
>gene1_sp2
atcg...
>gene2_sp1
atcg...
>gene2_sp2
Make sure bowtie2, spades.py, and samtools are callable in the current environment.
To assemble sequences in the target region, use the following command:
#Pair-end reads
python phyloherb.py -m assemb -r1 <Forward.fq> -r2 <Reverse.fq> -ref <reference fasta> -prefix <species ID> -n <threads>
#Single-end reads
python phyloherb.py -m assemb -rs <Single.fq> -ref <reference fasta> -prefix <species ID> -n <threads>
#Pair-end and Single-end, multiple libraries
python phyloherb.py -m assemb -r1 <lib1.R1.fq,lib2.R1.fq> -r2 <lib1.R2.fq,lib2.R2.fq> -rs <Single1.fq,Single2.fq> -ref <reference fasta> -prefix <species ID> -n <threads>
Ouput: In the working directory, PhyloHerb will generate prefix_spades folder, within which assembly scaffolds can be found.
Repeat this step for all species. The assembly step may run simultaneous and distributed to multiple jobs on a cluster.
Once the assembly is completed for all species, ortholog sequences can be scaffolded using the following command. The input dir should be the parent directory where all spades output folders are located. Make sure add the -nuc flag.
python phyloherb.py -m ortho -i <input dir> -o <output dir> -ref <reference.fasta> -nuc [optional] -n <threads> -evalue <evalue>
The default BLAST evalue is 1e-40. You can set more stringent values, but it is not recommended to use values larger than 1e-30.
If you have the assembly fasta files in one folder, add the -suffix flag to indicate file suffix:
python phyloherb.py -m ortho -i <input dir> -suffix <.fas/.fasta/etc> -o <output dir> -ref <reference.fasta> -nuc [optional] -n <threads> -evalue <evalue>
Ouput: In the output directory, there will be multiple *.fas files named after genes. Within each fasta file, the headers will be the species prefixes..
PhyloHerb will filter, select, and order BLAST hits for each reference gene. Below is an example from Cai et al (2022, unpublished), where a genome skimming assembly was mapped to a CDS reference, thus all BLAST hits were fragmented (likely exons). PhyloHerb will remove duplicates and select the optimum hit for each 'exon' based on evalues.
Raw BLAST result for loci R8408:
NODE_355_length_1022_cov_3.180816 R8408_Apios_americana_ge 91.246 297 23 1 616 912 754 1047 4.91e-117 417
NODE_79_length_1491_cov_2.984012 R8408_Apios_americana_ge 94.068 236 14 0 392 627 2171 2406 1.39e-100 363
NODE_2463_length_448_cov_3.594595 R8408_Apios_americana_ge 94.760 229 12 0 114 342 1283 1511 4.92e-100 360
NODE_4190_length_321_cov_2.393204 R8408_Apios_americana_ge 93.013 229 13 2 10 235 1511 1283 1.37e-92 334
NODE_355_length_1022_cov_3.180816 R8408_Apios_americana_ge 95.960 198 7 1 329 525 557 754 3.54e-87 318
NODE_2913_length_406_cov_2.250859 R8408_Apios_americana_ge 93.659 205 13 0 95 299 415 211 2.02e-85 311
NODE_2910_length_406_cov_3.487973 R8408_Apios_americana_ge 91.262 206 18 0 107 312 1508 1713 1.89e-79 291
NODE_2654_length_430_cov_4.333333 R8408_Apios_americana_ge 88.789 223 16 2 98 320 214 1 7.00e-79 289
NODE_1220_length_639_cov_2.805344 R8408_Apios_americana_ge 94.340 159 9 0 88 246 3256 3098 1.13e-65 246
NODE_79_length_1491_cov_2.984012 R8408_Apios_americana_ge 93.631 157 10 0 712 868 2399 2555 3.99e-63 239
NODE_3575_length_355_cov_2.200000 R8408_Apios_americana_ge 91.515 165 12 1 98 260 2047 1883 1.34e-61 232
NODE_79_length_1491_cov_2.984012 R8408_Apios_americana_ge 96.454 141 5 0 107 247 2032 2172 5.93e-61 232
NODE_4797_length_299_cov_1.750000 R8408_Apios_americana_ge 88.701 177 20 0 43 219 1712 1888 3.90e-61 230
NODE_1844_length_517_cov_3.522388 R8408_Apios_americana_ge 96.825 126 4 0 111 236 2794 2919 6.54e-55 210
NODE_3788_length_342_cov_1.295154 R8408_Apios_americana_ge 91.429 140 12 0 109 248 1287 1148 7.64e-52 199
NODE_79_length_1491_cov_2.984012 R8408_Apios_americana_ge 90.769 130 12 0 1058 1187 2554 2683 9.40e-46 181
NODE_355_length_1022_cov_3.180816 R8408_Apios_americana_ge 94.737 114 6 0 108 221 455 568 2.24e-45 179
NODE_1220_length_639_cov_2.805344 R8408_Apios_americana_ge 97.115 104 3 0 437 540 3100 2997 5.86e-44 174
Ordered and filtered BLAST hits by PhyloHerb, which will be used to assemble the full-length sequence of R8408 in the target species (Ordered by the last two columns):
qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore refstart refend
11 NODE_2654_length_430_cov_4.333333 R8408_Apios_americana_ge 88.789 223 16 2 98 320 214 1 7.000000e-79 289 1 214
9 NODE_2913_length_406_cov_2.250859 R8408_Apios_americana_ge 93.659 205 13 0 95 299 415 211 2.020000e-85 311 211 415
1 NODE_355_length_1022_cov_3.180816 R8408_Apios_americana_ge 95.960 198 7 1 329 525 557 754 3.540000e-87 318 557 754
18 NODE_355_length_1022_cov_3.180816 R8408_Apios_americana_ge 91.246 297 23 1 616 912 754 1047 4.910000e-117 417 754 1047
17 NODE_3788_length_342_cov_1.295154 R8408_Apios_americana_ge 91.429 140 12 0 109 248 1287 1148 7.640000e-52 199 1148 1287
7 NODE_2463_length_448_cov_3.594595 R8408_Apios_americana_ge 94.760 229 12 0 114 342 1283 1511 4.920000e-100 360 1283 1511
10 NODE_2910_length_406_cov_3.487973 R8408_Apios_americana_ge 91.262 206 18 0 107 312 1508 1713 1.890000e-79 291 1508 1713
15 NODE_4797_length_299_cov_1.750000 R8408_Apios_americana_ge 88.701 177 20 0 43 219 1712 1888 3.900000e-61 230 1712 1888
14 NODE_3575_length_355_cov_2.200000 R8408_Apios_americana_ge 91.515 165 12 1 98 260 2047 1883 1.340000e-61 232 1883 2047
4 NODE_79_length_1491_cov_2.984012 R8408_Apios_americana_ge 94.068 236 14 0 392 631 2171 2406 1.390000e-100 363 2171 2409
6 NODE_79_length_1491_cov_2.984012 R8408_Apios_americana_ge 90.769 130 12 0 1058 1187 2554 2683 9.400000e-46 181 2554 2683
16 NODE_1844_length_517_cov_3.522388 R8408_Apios_americana_ge 96.825 126 4 0 111 236 2794 2919 6.540000e-55 210 2794 2919
13 NODE_1220_length_639_cov_2.805344 R8408_Apios_americana_ge 97.115 104 3 0 437 540 3100 2997 5.860000e-44 174 2997 3100
12 NODE_1220_length_639_cov_2.805344 R8408_Apios_americana_ge 94.340 159 9 0 88 246 3256 3098 1.130000e-65 246 3098 3256
The final output sequence aligned to the reference is as follows:
>Apios_americana_ge(reference)
-----CTTCCTCGTGCAACTGTTGGTCCAGACGAGCCACATGCAGCAAGTACAACCTGGCCAGATGGTATTGCTGAAAAACAAGATTTAAGTGCAT---ATTCTGAACTA------ATAGAGGGGTTTCTAAGTTCTAAACTTCCATCTCACCCCAAGTTGCATCGAGGTCAACTGAAGAATGGGCTGCGTTATCTGATTTTGCCAAATAAAGTTCCACCAAAA---------AGGTTCGAAGCACACTTGGAAGTTCATGCAGGATCAATAGATGAGGCGGATGATGAGCAAGGAATTGCACATATGATTGAACATGTTGCTTTCCTAGGAAGTAAAAAACGTGAGAAGCTTTTGGGAACAGGAGCTCGTTCAAACGCTTATACTGATTTCCACCATACAGTGTTTCACATCCATGCTCCTACAAGCACCAAGGATTCTGATGATCTACTCCCATTTGTTCTGGATGCCTTGAATGAGATTGCTTTCCACCCAAAATTTCTTGCTTCTAGAATTGAAAAAGAACGGCGTGCTATACTCTCAGAGCTTCAAATGATGAATACAATAGAGTATCGAGTTGATTGCCAGTTGTTACAGCATCTGCATTCTGAAAATAAGCTGAGCAAAAGGTTTCCCATTGGATTAGAAGAACAGATAAAGAAGTGGGATGCAGATAAAATAAGGAAGTTTCATGAGCGTTGGTATTTCCCTGCAAATGCTACCTTGTACATTGTGGGGGATATTGATAACATCTCAAAGACTGTTTACCAGATTGAA------GCAGTTTTTGGACAAACTGGTGTAGACAATGGGAAAGGTTCTGTGGCCACTCCAAGTGCATTTGGTGCAATGGCTAGTTTTCTTGTTCCCAAGCTTTCTGTTGGTTTGGGTGCAAATTCTATTGAAAGATCAGCCAAT---ATGGATCAATCAAAAATATTCAATAAGGAAAGGCAAGCTGTTCGTCCTCCTGTGAAGCATAATTGGTCACTTCCTGGGAGTGGTGCTGATTTGAAACCACCACAAATATTTCAACACGAGTTACTTCAAAATTTTTCAATTAATATGTTCTGCAAGATTCCAGTGAATAAGGTTCAAACATACAGAGACTTGCGCTTGGTCTTGATGAAAAGAATATTTTTGTCTGCTCTTCATTTTCGAATTAATACAAGATATAAGAGTTCAAATCCACCATTCACTTCAGTCGAATTGGATCACAGTGACTCTGGTAGGGAAGGATGCACCGTGACCACTCTTACCATAACTGCAGAACCAAAGAATTGGCGAAATGCAATTAGAGTTGCTGTTCAAG----------AGGTTCGGAGACTTAAAGAGTTTGGAGTTACTCAGGGTGAATTAACTCGTTATTTAGATGCCCTTCTTAAAGATAGTGAACACCTAGCAGCCATGATTGATAATGTATCTTCTGTTGATAACTTGGATTTTATCATGGAAAGTGATGCTCTTGGCCATAGAGTTATGGACCAGAGACAAGGGCATGAAAGTTTACTTGCTGTTGCTGGGACAGTTACCCTTGAGG---------AGGTTAATTCTGTTGGTGCCAAGGTGTTAGAATTTATAGCTGATTTTGGAAAGGCTACTGCACCACTTCCTGCAGCAATTGTTGCTTGTGTTCCAAAAAAAGTTCACATTGAGGGATCTGGTGAAACAGAATTCAAGATATTATCAACTGAAATTACGGATGCTATGAAAGCTGGGTTGGATGAACCTATTGAGCCAGAGCCTG-------AGCTTGAGGTGCCAAAAGATCTGATACAATCATCAAATCTAGAAGAGTTGAAAACCCTGCGCAAGGTGGCCTTTATTCCTGTAAATCCTGAAACAGATGCTACAAAGCTTCATGATGAGGAAACGGGGATCACCCGGCGCCGTCTTTCAAATGGAATTCCTGTTAATTATA-----------AGATATCAAAAACTGAAACACAAAGTGGTGTAATGCGGCTGATTGTTGGTGGTGGACGGGCAGCTGAGAGTTCTGAGTCAAGAGGATCTGTGATTGTGGGTGTTAGGACACTTAGTGAGGGAGGTCGTGTTGGCAACTTCTCAAGGGAGCAGGTGGAACTTTTCTGTGTAAATCACCTGATAAATTGCTCCTTGGAATCTACGGAGGAATTCATATCCATGGAGTTCCGTTTTACTTTAAGAGACAATGGGATGCGTGCGGCCTTTCAATTGCTTCACATGGTGCTCGAGCATAGTGTCTGGGTTGACGATGCTTTCGATAGAGCAAGGCAATTGTATCTGTCATATTACCGATCCATCCCCAAGAGCTTGGAACGCTCAACCGCACACAAACTCATGGTAGCAATGCTGGATGGGGACGAGCGGTTCATTGAGCCTACACCAAAATCACTAGAAAATTTAACCCTGCAATCTGTTAAGAATGCAGTAATGAATCAATTTGTTGGTGATAACATGGAGGTATGCATTGTAGGTGACTTCACTGAGGAGGACATTGAGTCTTGCATTCTTGATTACCTTGGCACAGCTCAGGCCACAAGAAATCACCAAAGTGAACAAGAATTCAACCCACCCTTATTTCGACCATCTCCATCTGATTTGCAGTTTCAAGAAGTATTTTTAAAGGACACCGATGAGAGGGCATGTGCTTATATAGCTGGACCAGCGCCAAACCGTTGGGGTTTTACTGTTGATGGAAAAGACTTGTTAGAGTCAATTAATAATGCATCAACAATCAA----TGATGATCCGTCAAAATCTGATACCCAACAGACCGGTGGTTTGCAAAAGAGCCTTCGTGGTCATCCTCTTCTCTTTGGTATAACAATGGGACTGCTTTCCGAGATTATAAATTCTAGGCTCTTCACAACTGTCAGAGATTCTCTGGGATTGACATATGATGTGTCATTTGAATTAAACTTGTTCGATAGGCTTAAACTAGGATGGTATGTGATCTCTGTGACATCAACTCCAAGCAAGGTGCACAAAGCTGTTGATGCATGCAAGAATGTTCTTAGGGGTCTGCATAGCAACAAAATTACTGAGAGGGAATTGGACAGGGCTAAACGGACCCTTCTTATGAGACATGAAGCTGAAATTAAGTCAAATGCCTACTGGCTAGGATTATTAGCTCACTTACAAGCTTCTTCTGTTCCAAGGA--------AGGACATATCATGTATCAAGGACCTAACATTTCTATATGAAGCTGCTACTATTGAGGATATATACCTTGCATATGAACAATTGAAAGTGGATGAGAATTCTCTATATTCATGCATTGGAATTGCTGGTGCTCAGGATGCACAAGATATAGCAGTTGAAGAGGAAGATGCTTATCCAGGTGTTATTCCGGTGAGAGGTTTATCTACGATGACACGGCCAACTACC
>T66-3(target)
NNNNNCTTCCACATGCAACTGTTGGTCCAGATGAGCCACATGCAGCAAGTACGACCTGGCCAGATGGTATAGCTGATAAGCAAGATTTAAGTGTATTTGATTCTGAACTAGAGCGGATAGAGGGGTTTTTAAGTTCTGAACTTCCGTGTCACCCTAAGTTGTATCGAGGTCAACTAAAGAATGGGCTGCGTTATCTGATTCTGCCAAATAAAGTTCCACCAAAAAGGTNNNNNAGGTTTGAAGCACACTTGGAAGTTCATGCAGGATCAATAGATGAAGAGGATGATGAGCAAGGCATTGCACATATGATTGAACATGTTGCTTTCTTAGGAAGTAAAAAACGTGAGAAGCTTCTGGGGACAGGAGCCCGTTCAAATGCTTATACTGATTTTCACCATACAGTGTTTCACATCCATGCTCCTACCAGTACCAAG---------------------------------------------------------------------GTTTNNNNNATTG--------------------------------------------------------------------CAGTTGTTACAGCATCTGCATTCTGAAAACAAGCTGAGCAAAAGGTTTCCAATTGGATTAGAGGAACAGATAAAGATATGGGATGCAGATAAAATAAGGAAATTTCATGAGCGTTGGTATTTCCCTGCAAATGCTACCTTGTACATTGTGGGGGATATTGATAACATCCCAAAGACTGTTTACCAGATTGAAGNNNNNGCTGTTTTTGGACAAACCGGTGTAGACAATGAGAAGGGTTCTGTACCCACTCCAAGTGCATTTGGCGCAATGGCTAGTTTTCTCGTTCCTAAGCTCTCTGTTGGTCTGGGTGGAAATTCTATTGAAAGATCAGCCAATACAATAGATCAATCAAAAATATTCAGTAAGGAAAGGCAAGCTGTTCGTCCTCCCGTGAAGCATAATTGGTCACTTCCTGGAAGCAGTGCGGATTTGAAGCCACCACAAATATTTCAGCATGAGTTGCTTCAAAATTTTTCAATTAATATGTTCTGCAAGNNNNN-----------------------------------------------------------------------------------------------AGAGTTCAAATCCACCATTCACTTCTGTTGAATTGGATCATAGTGATTCTGGAAGGGAAGGATGTACTGTGACCACTCTTACCATAACTGCAGAACCGAAAAATTGGCAAAGTGCAATTAGAGTTGCTGTTCATGAGGTTNNNNNAGGTTCGGAGACTTAAAGAGTTTGGTGTTACCCAGGGTGAATTAACTCGTTATTTAGATGCCCTTCTAAAAGATAGTGAACACCTAGCAGCCATGATTGATAATGTGTCTTCTGTTGATAATCTGGATTTTATCATGGAAAGTGATGCTCTAGGCCACAAAGTTATGGACCAGAGACAGGGGCATGAAAGTTTACTCGCAGTTGCTGGGACAGTTACCCTTGAGGAGGTNNNNNAGGTCAATTCTGTTGGTGCCAATGTGTTAGAGTTTATAGCTGGTTTTGGAAAGCCTACTGCACCCCTTCCTGCAGCAATTGTTGCTTGTGTTCCGAAAAAAGTTCACATTGAGGGAACCAGCGAAACAGAATTCAAGATATCATCAACTGAAATTACAGATGCTATCAAAGCTGGATTTGATCAACCTATTGAGCCTGAGCCTGAGNNNNNAGCTTGAAGTGCCAAAAGAACTGATACAATCATCGAAGCTAGAAGAGTTAAAAATGCAGCGCAAGCCAGCCTTTATTCCGGTAAGTCCTGAATCAGAAGCTACAAAGCTTCATGACGAGGAAACCGGGATCACCCGGCGCCGTCTTGCAAATGGAATTCCTATTAATTATAAGGTATNNNNNAGATATCAAAAACTGAATCACAAAGCGGTGTGATGCGATTGATTGTTGGTGGCGGACGGGCAGCTGAGAGTTCCAAGTCAAGAGGATCTGTGATCGTGGGTGTTAGGACGCTTAGTGAGGGAGGCCGTGTTGGCAACTTCTCAAGGGAGCAGGTT--ACTTTTC----------------------------------------------------------------------------------------------------------------------TNNNNNAGCATAGTGTCTGGGTAGATGATGCTTTTGATAGAGCAAGGCAATTGTATCTGTCATATTATCGATCCATCCCCAAGAGCTTGGAACGCTCAACTGCTCACAAACTAATGGTAGCAATGTTGGATGGAGACGAGCGATTTATTGAGCCTACACCAAAATCACTAGAAAATTTAACTCTGCAATCTGTTAAGGATGCAGTAATGAATCAATTTGTTGGTGATAACATGGAGGTCTGC------------------------------------------------------------------------------------------------------------------------------------------TATTNNNNNGTATTTTTAAAGGACACCGACGAGAGAGCATGTGCATATATTGCTGGGCCAGCACCAAACCGTTGGGGTTTTACTGTTGATGGAGAAGACCTGTTAGTGTCAATTAATAATGCATTAACAATCAG----TGGTGNNNNN---------------------------------------------------------------------------------------------------------AGGCTCTTCACAACTGTCAGAGATTCACTGGGGTTGACATATGATGTGTCATTTGAATTAAACTTGTTTGATAGGCTTAAACTAGGATGGTATGTGATCTCTGTGACATCAACTCCGAGCAAGG------------------------------------------------------------------------TGNNNNNGGCTAAGCGGACCCTTCTTATGAGACATGAAGCTGAAATTAAGTCAAATGCCTACTGGCTGGGATTGTTAGCTCACTTACAAGCTTCTTCTGTTCCAAGGAAGGNNNNNAGGACATATCATGTATCAAGGACCTAACATTTCTATATGAAGATGCTACTATTGAGGATATATACCTTGCATATGAACAGTTGAAAGTGGATGAAAATTCTCTGTATTCATGCATTGGGATTGCTGGTGCTCAGGCTGCACAAGATATTGCAGGTGCAG-----------------------------------------------------------------
Overview
If interested in phylogeny alone, up to 384 samples (4 plates * 96 samples/plate) can be multiplexed on a single Illumina HiSeq 2500 lane for most flowering plants. Using the NovaSeq plastform can generate more complete genomes thanks to its larger output, but currently we cannot put more than 384 multiplexed samples due to the barcode limitation. If circularized plastid genomes are needed, >2 Gb data per species can usually get you there, which translates to ~60 samples per lane.
IMPORTANT: If your species have fewer-than-usual plastids per cell or exceptionally large genomes, you need to reduce the number of multiplexed species per sequencing lane. Use the following equation to calculate the expected base coverage of plastid genome:
Minimally, you want the plastid coverage to be larger than 10X.
FAQ
- DNA extraction from herbarium specimens? How?!
We have successfully extracted DNAs from 200-year-old specimens. Age matters less than the preservation methods (see this paper). Standard commercial DNA extraction kits are frequently used to obtain DNA (e.g, Tiangen DNAsecure Plant Kit, Qiagen DNeasy Plant Mini Kit). We used a Promega Maxwell instrument that can extract DNA from 16 samples simultaneously within an hour. This automatic approach is certainly more labour efficient, but manual extractions have more guaranteed yields for delicate samples.
- Where can I find the genome sizes of my species?
In addition to searching through the literature or conducting your own flow cytometry experiments, you could also check the Plant DNA C-value database organized by Kew.
- NGS library preparation and multiplexing
We used the KAPA HyperPlus Kit for NGS library. Many institutes provided services for NGS library preparation with robots. We have used quarter reaction (1/4 of all reagents) for our NGS libraries, and it works just fine.
- Where are the limits?
About 0.5-6% of the reads from genome skimming come from plastomes. The base coverage is roughly half for mitochondria and 2X for nuclear ribosomal regions compared to plastids. Theoretically the base coverage vary with the size of the nuclear genome and the abundance of these genetic regions within a cell, but we found it to be relatively consistent across flowering plant species despite the dramatic difference in their genome sizes (200 Mb to 3Gb). Below is a very rough estimation of what you may expect for plastome from certain amount of input data.
