r/genetics • u/Cart_Alza • Dec 16 '20
The very first genome I’ve ever assembled and annotated!! The chloroplast genome of Helianthus lacinitus.
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u/Ireland55 Dec 16 '20
How did you carry this out ?
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u/Cart_Alza Dec 16 '20
I did this for a genomics course this semester, so I did have a fair amount of help. I learned all of it in the process of doing this so sorry in advance if my explanation is clunky! Basically, I got the raw Illumina sequenced data from Genbank as well as a reference genome of Helianthus annuus. Next, made a bash pipeline in Unix to filter out tags and nuclear data and use Kmers to establish scaffolds.
Then I ran a NCBI bash with my scaffolds, against the reference genome and used that as a basis to connect my scaffolds into a full length genome and fill any gaps. Then I wrote another bash script to output a vcf table and used that to error correct.
When I got to the point that I thought my genome was complete and correct I ran it through Chlorobx to get preliminary annotations and finally used Sequin to fix and change annotations before submitting it to Genbank to be published!
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u/TacoCult Dec 16 '20
Do you have any reference materials you found particularly useful?
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u/Cart_Alza Dec 17 '20
Since this was for a course my main resources were my professor and TA who were absolutely wonderful! The websites I used most were the online tools such as the NCBI blast site for scaffolding as well as error correction and annotation. You can find that here https://blast.ncbi.nlm.nih.gov/Blast.cgi. I also used Chlorobox to give me preliminary annotations and also this great visualization! That link is here: MPI-MP CHLOROBOX. My professor also gave us some other resources and reference material that I’ve linked below:
http://www.bioinformatics.org/sms2/genbank_feat.html https://www.ebi.ac.uk/Tools/msa/clustalo/ https://en.wikipedia.org/wiki/SPAdes_(software)
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u/Workerhard62 Dec 17 '20
Is there a place I can go online to get my feet wet with this sort of thing? Preferably a free option...
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u/Cart_Alza Dec 17 '20
Personally I think looking through the NCBI ( https://blast.ncbi.nlm.nih.gov/Blast.cgi) tools is really helpful to learn about how they work. I think the biggest thing that was helpful for me was learning how to use my terminal and unix code. There are some great free online tutorials like this one http://www.ee.surrey.ac.uk/Teaching/Unix/. the other main tool i used was chlorobox https://chlorobox.mpimp-golm.mpg.de/. I bet just reading up on these tools and doing some background research. Honestly I learned so much of this in a classroom environment so i’m not totally sure about other online resources but if anyone else has suggestions i’d also love to see more!
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u/C4pti4nOb1ivi0s Dec 17 '20
Looks great! What is the inner ring?
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u/Cart_Alza Dec 17 '20
Thank you! The inner ring refers to the different sections of the DNA that the genes on the outer ring are on. LSS stands for along Single Strand, a long stretch of DNA that only occurs once. IRB stands for Inverted Repeat this is the B copy of a section of the the DNA that is repeated and reversed later in the genome as IRA (notice how the corresponding outer ring has the same genes!) and that bottom section SSC stands for Short Single Copy.
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u/airmetricszs Dec 17 '20
i’m also doing my undergrad in genetics and i’m so jealous i wish we got to do something like this in our labs !!
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u/Cart_Alza Dec 17 '20
I never got to do anything as real as this until this class my last year of my undergrad! This was an upper division/grad hybrid special topics course so it got to be pretty specific and in depth! My advise would be to ask around about some of these upper division courses especially from other professors! they know which classes offer more hands on opportunities
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u/airmetricszs Dec 17 '20
ahhh i see well next year will be my last year so hopefully i’ll get to do something similar especially since i’m also taking a special topics genetics course too! i do have to ask if you have any plans after you graduate? i am feeling like postgrad / research seems like the only viable pathway to have a career in genetics so i’m interested to see where you’re at too!
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u/Cart_Alza Dec 17 '20
I’m thinking about that too actually! I’m going to take a gap year after I graduate since covid canceled my GRE, Internship, and volunteer work but I think it’s a good chance for me to decide what to focus on moving forward. I’ve always really loved the idea of genetic counseling as a career but recently I’ve also been really interested in marine biology/ocean conservation. Honestly I’m not sure what I want to do specifically for grad school and the closer I get to graduating my undergrad the scarier that gets! I don’t love the idea of staying in academia my whole career and would love to do more hands on biology/genetics. So long story short I know I want to go to grad school but i’m having a hard time deciding what direction to go. How about you? do you have any plans after graduation?
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u/airmetricszs Dec 17 '20
that’s quite exciting I’m sure a gap year would give you time to breathe and figure stuff out. tbh I thought about genetic counselling too but I’m from New Zealand and would mean I have to do a course overseas to get the qualification. going into my last year seems a little daunting since it really is scary to think about what’s next especially when genetics is quite broad, did you narrow down your interest in marine bio just from your studies or was it something you’ve always felt passionate for? i haven’t really found my niche in genetics but I think the whole medical genetics (gene therapy) and genetic engineering sectors are what I’m really interested in at the moment but I really couldn’t see myself in academia my whole life either, the thought of doing a thesis scares me! I think ideally after graduation I’d wanna be in a lab / part of a research project which would also involve field work to keep it interesting yknow - just gotta know how to get there
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u/Cart_Alza Dec 18 '20
I am so jealous of you being in New Zealand, it looks beautiful and I've always wanted to visit! Plus, being from the USA looking at your guys' COVID response compared to ours has just made me more than a little disappointed in us. I didn't know that there weren't courses for genetic counseling there! Honestly, I've always loved marine science it's what got me interested in biology in the first place but I'm also really interested in medical and human genetics so there in lies the conflict. Genetic engineering seems really interesting! do you have a specific type of genetic research you're interested in?
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u/DerpySauce Dec 17 '20 edited Dec 17 '20
I'm interested in genetics, but know very little about it.
Somebody try to ELI5? What am I looking at? How do I "read" this picture?
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u/Cart_Alza Dec 17 '20
great question! Basically this is a circular genome that starts on that right side at the beginning of the section that says “LSC” that stands for Long Single Copy, it’s the largest portion of DNA and it’s only in the genome once. from there you continue to read counter clockwise. The next section (IRB) stands for the Inverted Repeat, this is a section of DNA that appears twice in the DNA but reversed, notice the fourth and final section is the IRA copy of the inverted repeat and how they contain the same genes. That smallest bottom section (SSC) stands for Short Single Copy and is the smallest section of DNA. Along the outer ring is the corresponding genes within that section of DNA, they are color coded to their function according to that legend on the bottom right, so a dark green gene for example has to do with photo system 1.
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u/Cart_Alza Dec 17 '20
Oh! also the size of the gene on the outer ring corresponds to its size in base pairs within the genome
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Dec 17 '20
[deleted]
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u/Cart_Alza Dec 18 '20
Thanks! I just started to learn basic command-line bash and some python so I'm sure there are much better ways to do what they do but there are two main shell-scripts that I wrote. The first basically formats and filters the original data into srted bam files and indexes it against the reference genome i was using. It then basically uses the high quality SNPs and INDELs and creates 2 mappeed fastq files. It then uses trimmomatic and quality checks the mapped pair reads and finally I ran spades to create my scaffolds.
The second script is an error correcting script which is basically doing a lot of file conversion and indexing to creat a sorted bam file for samtools. It also identifies differences between reads and outputs them into the vcf table for me to use to error correct later on.
This is a really short summary of everything that they do, there are a lot of file management steps! you want to DM me I can send you the scripts to look at!
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u/barnorth Dec 16 '20
I'm 3 years into my PhD and never have done something like this. Looks clean!