Hi,
I'm doing single-cell TCR sequencing using Mark Davis' protocol. Here is the link to Davis paper: http://www.nature.com/nbt/journal/v32/n7/abs/nbt.2938.html
The paper used vdjfasta for TCR analysis. But the vdjfasta was set up for antibody sequence analysis. When I use it for TCR sequences, I don't get exactly what described in the paper. Could anyone help me? I wonder if anyone can tell me how to change vdjfasta (probably the database or hmm file?) for TCR analysis or point me to another tool.
Thank you so much in advance!
Bin
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written
by
binfeng •
2 answers
Hello!
I can recommend one of our tools, MIGEC/CdrBlast or MITCR. The latter is slightly less accurate but much faster.
Here is the full list of immune repertoire sequencing analysis tools: http://omictools.com/rep-seq-c424-p1.html
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written
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mikhail.shugay
A real-world example would be quite large as it need to cover molecular tag (~one cDNA molecule, see original paper) 10-20 times for each of several clonotypes. You can still use the full dataset, see http://www.ncbi.nlm.nih.gov/sra?term=PRJNA239303 .
I've just uploaded a single sample from http://www.jimmunol.org/content/early/2015/05/08/jimmunol.1500215 with TCR beta to S3, feel free to download the read1 and read2 files, they contain the unique molecular tag sequence in header in case you need it.
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written
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mikhail.shugay
I just downloaded read1 as testing input file. It worked! But I still have a couple of questions. Here is the command I used:
java -jar migec-1.2.1a.jar CdrBlast -R TRB S2-1-beta_R1.fastq.gz r1.out.txt
My questions are:
- In the output file (r1.out.txt), it contains all the information I need except identifiers for each TCR. In other words, there is no UMI information in the output file. Did I use your tool correctly?
- Should I use Checkout and how do I use it?
Thank you so much!
Bin
Dear Bin,
Sorry for a late reply.
You do not need to use Checkout, they are already split by sample barcodes and the info is already in read header.
You need to run Assemble to assemble consensuses and then CdrBlast. It will process the UMI info from header.
You don't need those barcodes as files are already de-multiplexed with adapter sequences containing sample barcode cropped. Still, if you download raw files from SRA you can use them. Here are barcodes for alpha and beta chains:
I would be very interested on your feedback on the tool I wrote, clonotypeR. But note that for the moment it misses a data file for human TCR analysis (draft instructions are available to make one.)
Indeed, that was not the best link to start with; sorry. Have a look at http://clonotyper.branchable.com/references/README/. It briefly summarises how I prepared the reference alignments for mouse. Basically, you need to align the DNA sequence of the V segments on the conserved cysteine and the DNA sequence of the J segments on the FGxG motif. I could only do it by hand (for instance, for some V segments the conserved cysteine is not the last one), and I found that SeaView was the best tool for this.
Also, if you contact me by email, I can send you the draft paper that I wrote to describe clonotypeR more in details.
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Charles Plessy
Hi Bin,
I am a bioinformatician in the cancer immunotherapy lab at Duke university and have just started working on a single cell TCR sequencing project. I was also trying to follow the Mark Davis paper that you referenced. It seems they had modified vdjfasta for TCR but I am not sure if the modified version is available. It would be very helpful if you could share your experience and provide me some information on what tools you used for your analysis and any details related to the analysis. Also, do you know if the raw data from the Mark Davis is available or not. I have written to him to find out but have not heard back yet.
Thanks,
- Pankaj
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written
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bioinformatics.cancer
Hi, I would recommend using MiXCR for the repertoire data, as it has a specific command designed to work with libary structure from this paper:
mixcr analyze han-et-al-2014-bcr \
--species hsa \
input_R1.fastq.gz \
input_R2.fastq.gz \
result
Using that command you can easily get the results for repertoire data analysis.
You can read more about this here:
https://docs.milaboratories.com/mixcr/reference/overview-built-in-presets/#han-et-al-2014
Also, feel free to contact us on github if you have any questions or suggestions: https://github.com/milaboratory/mixcr
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