making-multigene-family-maps-from-long-read-genomes

These scripts can help you get from a set of long-read contigs and blast hits to a nice figure of your multigene family

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Making quick maps of multigene families using blast, genome assemblies and gggenes

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knitr::opts_chunk$set(echo = TRUE)

Introduction

This module will help you go from a set of long-read genome contigs to a simple representation of a complex multigene family like the major histocompatibility complex. This is not a substitute for full-on annotation, but it may be useful if you want a quick overview of a multigene family across several haplotypes of the same species. We will use the scrub jay MHC as an example and we'll assume that you have already generated formatted tables of blast hits using the command-line blast. For this you will need to setup a blast database of your genome to be blasted, as well as find the appropriate query sequences of genes you are interested in representing. I have found these resources to be helpful in doing this: https://www.ncbi.nlm.nih.gov/books/NBK569856/ and https://www.metagenomics.wiki/tools/blast/blastn-output-format-6. For example, let's imagine I have several genomes (fasta files) ending with the suffice "ren". On the Cannon cluster, you can run the command:

module load blast/2.6.0+-fasrc01
for file in *ren*;
  do makeblastdb -in "$file" -dbtype nucl -out ${file%.*};done

You can then make an 'uber' database of all your genomes (in this case, the "MCZ" files) with this command:

blastdb_aliastool -dblist "MCZ_orn_365326.hap1.fa_ren MCZ_orn_365326.hap2.fa_ren MCZ_orn_365338.hap1.fa_ren MCZ_orn_365338.hap2.fa_ren \
MCZ_orn_366490.hap1.fa_ren MCZ_orn_366490.hap2.fa_ren MCZ_orn_366493.hap1.fa_ren MCZ_orn_366493.hap2.fa_ren MCZ_orn_366494.hap1.fa_ren MCZ_orn_366494.hap2.fa_ren" \
-dbtype nucl -out scrubjay_9_haps -title "10 scrub jay renamed genomes combined"

Getting your blast output together

Now by blasting to this library (called "scrubjay_9_haps") you can blast to all the individual genome databases simultaneously. You will want to set your output format when you blast so that it generates a table that is easily manipulated. I did this by using the -outfmt "6" command, further specifying the precise columns to generate using the codes available here. So the commmand to generate a table for mhc class II exon 3 using part of this scrub jay sequence as a probe might look like this:

blastn -db scrubjay_26_haps -query Mhc_U23958.1_exon3.fa -out sj_mhc2_exon3_blast.out -task megablast -outfmt "6 qseqid sseqid pident sstart send sstrand"

Your output should be a 6 column table of blast hits and target sequence ids. You may want to add additional columns but we will mainly use qseqid, sseqid, sstart and send. Let's retrieve that file, as well as other gene blast tables we might use as anchors in our map, and look at them:

Preparing the blast outputs in R

library(ggplot2)
library(tidyverse)
library(ggfittext)
library(gggenes)
library(RColorBrewer)
setwd("/Users/scott/OneDrive\ -\ Harvard\ University/Research/Scrub\ Jays/26_haps/blasts/gggenes\ layouts")
mhc3<-read.table("sj_mhc2_exon3_blast.out")
brd2<-read.table("sj_zfbrd2_26_blast.out")
slc39<-read.table("sj_slc39A7_26_blast.out")
dim(mhc3)
head(mhc3)

Let's put the gene (query) name in the first column of each table for clarity:

mhc3[,1]<-"MHC_classIIB_exon3"
brd2[,1]<-"BRD2" 
slc39[,1]<-"SLC39A7"

Subsetting the scaffolds

Now let's find out which scaffolds have all three of the genes we are interested in, join the tables together and re-name the tables. The second column (V2) of each table has the scaffold hits:

corescaffs<-intersect(mhc3$V2,union(brd2$V2,slc39$V2))
corescaffs
fulltab<-rbind(mhc3,brd2,slc39)
head(fulltab)
colnames(fulltab)<-c("gene","molecule","perc_ident","start","end","strand")
intertab<-fulltab %>% filter(molecule %in% corescaffs)

Now we have a table of scaffolds that at least have an MHC hit as well as a BRD2 or SLC39 hit. There are a total of 26 unique contigs/scaffolds in this table. The single-copy gene BRD2 in particular is a good anchor gene. SLC39 and RXRB are also good anchor genes, even if SLC39 and RXRB are small multigene families. Exon 4 of RXRB is a good low-copy conserved probe to delimit the passerine MHC class II region.

Putting contigs/scaffolds in a common orientation

I find it helpful now to determine the orientation of genes on each scaffold, since we want to display them all in the same orientation. I do this by making a table of hits ordered by position in scaffold, and then asking if the BRD2 is at the beginning or end of the scaffold (beginning or end is arbitrary). I want to identify scaffolds with BRD2 at the end, so I can reverse them, because I want BRD2 to be on the left end of each of my maps.

scaffs<-unique(intertab$molecule)
for(i in 1:length(scaffs)){
  list<-intertab %>% filter(molecule == scaffs[i]) %>% arrange(start)
  write.table(list,"scaff_arrange_tab.txt",sep = "\t",append = T,col.names = F,quote = F,row.names = F)
}

By inspecting the file scaff_arrange_tab.txt I found that 15 of the 26 scaffolds needed reversing. I put the names of these scaffolds in a list called strands.

strands<-c("MCZ_Orn_366498.hap2.h2tg000086l","MCZ_Orn_366498.hap1.h1tg000659l","MCZ_Orn_366487.hap2.h2tg000648l",
           "MCZ_Orn_365336.hap1.h1tg000632l","MCZ_Orn_365335.hap2.h2tg000459l","MCZ_orn_365326.hap2.h2tg000420l",
           "MCZ_orn_365326.hap1.h1tg000679l","MCZ_orn_366494.hap1.h1tg000827l","MCZ_orn_366493.hap2.h2tg000471l",
           "MCZ_orn_366493.hap1.h1tg000074l","MCZ_orn_366490.hap1.h1tg000470l","MCZ_Orn_366499.hap2.h2tg000716l",
           "MCZ_Orn_366488.hap2.h2tg000702l","MCZ_Orn_365337.hap1.h1tg000802l","MCZ_Orn_365327.hap2.h2tg000373l")

Now let's create new columns to either reverse or preserve the orientation of the start and end of blast hits, as appropriate:

`%notin%` <- Negate(`%in%`)
negall<-intertab[which(intertab$molecule %in% strands),]
notreverse<-intertab %>% filter(molecule %notin% strands)
reverseall<-mutate(negall,negstart = -start,negend = -end)
notreverse<-mutate(notreverse,negstart = start,negend = end)
reversetab2<-bind_rows(reverseall,notreverse)

We can inspect the order of genes on individual scaffolds now. For example:

reversetab2 %>% filter(molecule == "MCZ_orn_366494.hap1.h1tg000827l") %>% arrange(start)

Some of the genes have negative positions, but that's ok. The fact that BRD2 has the lowest position (most negative) assures me that it will be on the left of the map when I plot it.

Making small genes on large contigs a bit easier to see

Now we need to find out which direction each gene is in so that we can add a bit more length to it in the appropriate orientation so that we can see individual genes better. This example involves contigs that are about 400 kb in length; by adding 3kb to the beginning and end of each gene we can see it better but we must remember that our diagram is now not strictly to scale.

norm2<-which(reversetab2$negstart < reversetab2$negend)
reversetab3<-mutate(reversetab2[norm2,],negstart03=negstart-3000,negend03=negend+3000)
reversetab4<-mutate(reversetab2[-norm2,],negstart03=negstart+3000,negend03=negend-3000)
reversetab5<-bind_rows(reversetab3,reversetab4)

Anchoring your scaffolds on a gene and common scale

Now let's decide to anchor our diagram on the BRD2 gene. We do this by constructing a dummy alignment using functions in gggenes and the columns indicating our expanded gene hit sizes. When we do this, we then need to focus only on those scaffolds that actually have the BRD2 gene. This will lower our scaffold number from 26 to 19.

dummies <- make_alignment_dummies(
  reversetab5,
  aes(xmin = negstart03, xmax = negend03, y = molecule, id = gene),
  on = "BRD2"
)

Now, in order to plot each haplotype on a common axis, we need to do some magic that I learned from David Wilcox, author of gggenes, who kindly answered some questions for me on his github site. Basically we are re-setting each gene on a common scale.

reversetab5 <- reversetab5 %>%
  group_by(molecule) %>%
  mutate(genes_min = min(c(negstart03, negend03))) %>%
  mutate(negstart03 = negstart03 - genes_min) %>%
  mutate(negend03 = negend03 - genes_min) %>%
  ungroup()

Plotting!

Finally, let's compile all the scaffolds that have the BRD2 gene and then plot just those.

brd2scaffs<-(reversetab5 %>% filter(gene == "BRD2"))$molecule

It will work better to make your R studio plot window large before running this chunk. If you get an error like "Error in grid.Call..." it probably means your R studio window is too small. Better to pass the plot to a variable and then use ggsave with a large output to save it. You can vary the thickness of the gene maps, the colors of the genes, and many other aspects using parameters in this scipt. Look what a nice figure you've made!

reversetab5 %>% filter(molecule %in% brd2scaffs) %>% ggplot(aes(xmin = negstart03, xmax = negend03, y = molecule, fill = gene, label = gene)) + 
  geom_gene_arrow(arrowhead_height = unit(3, "mm"), arrowhead_width = unit(1, "mm"), 
                  colour = "blue", size = 0) +
  facet_wrap(~ molecule, scales = "free", ncol = 1) +
  scale_fill_brewer(palette = "Set3") + 
  scale_x_continuous(labels = scales::comma, limits = c(0, 420000)) + 
  theme_genes() %+replace% theme(axis.text = element_text(size = 10))

or

p<-reversetab5 %>% filter(molecule %in% brd2scaffs) %>% ggplot(aes(xmin = negstart03, xmax = negend03, y = molecule, fill = gene, label = gene)) + 
  geom_gene_arrow(arrowhead_height = unit(3, "mm"), arrowhead_width = unit(1, "mm"), 
                  colour = "blue", size = 0) +
  facet_wrap(~ molecule, scales = "free", ncol = 1) +
  scale_fill_brewer(palette = "Set3") + 
  scale_x_continuous(labels = scales::comma, limits = c(0, 420000)) + 
  theme_genes() %+replace% theme(axis.text = element_text(size = 10))

ggsave("Mhc_classII_gene_plot.pdf",p, height = 20, width = 10,limitsize = FALSE)

There are also ways to remove all but the top or bottom scale, although at this stage I just remove the unwanted scales in Illustrator.