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I have been running nf-core in Python and it works great! But I have a seemingly simple question that I'm struggling to find an answer for online. After running the nf-core pipeline on my RNA sequencing data, Salmon is able to provide gene counts in a file named salmon.merged.gene_counts.tsv. This file has essentially all the information I am seeking with all counts associated to a unique gene_id. But, for a large number of these gene_ids where no specific gene_name is known, I would like to further find the corresponding DNA sequence (or corresponding transcript's RNA sequence) for each gene_id. Accordingly, my question is: where can I find the information to map the gene_id to the corresponding nucleotides of the gene's (or original transcript's) sequence?

(Apologies in advance if my question is missing information or overly simply and I'm overlooking something obvious — I am quite new to this all.)

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  • $\begingroup$ Which species? Also:can you provide any examples of gene_ids you have problems with? $\endgroup$
    – darked89
    Commented Feb 15 at 12:18
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    $\begingroup$ Anas platyrhynchos (mallard) — I'm trying to actually do the inverse problem and check if TP53 is present as one of the gene_ids. Looking at related data from https://midb.pnb.uconn.edu/, this gene seems to be missing there as well $\endgroup$
    – user18959
    Commented Feb 15 at 12:33
  • $\begingroup$ re TP53: if one search for p53 gene here: ensembl.org/Anas_platyrhynchos/Info/Index I do not see the TP53 itself, just the genes binding to it, or downstream targets. If the genome is incomplete/full of gaps then tp53 may be missing from annotation. I guess not just in ENSEMBL but from other genome assembly based as well. $\endgroup$
    – darked89
    Commented Feb 15 at 12:59
  • $\begingroup$ If you look at the orthologues of human TP53 (ENSEMBL), you can find them in chicken, Kakapo, Zebra finch plus a bunch of reptiles. While this is not an ultimate proof that TP53 does exist in your duck, I would expect that some duck RNA-Seq may contain at least fragments of TP53. $\endgroup$
    – darked89
    Commented Feb 15 at 13:26
  • $\begingroup$ Thank you for sharing these thoughts, @darked89. I suppose my simple question is: what is the best way to check the duck RNAseq data for any fragments of TP53? Any specific insights on methods and files to utilize would be helpful! $\endgroup$
    – user18959
    Commented Feb 15 at 23:29

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fishing out tp53 RNA-Seq reads

  • go to NCBI Taxonomy Browser
  • put Anas platyrhynchos as a search term
  • click on Anas platyrhynchos
  • on the right you will have a table with SRA Experiments, click on 11,679
  • restrict to RNA and paired on the left
  • send it to Run Selector
  • order it by the Bases
  • select few top runs, like SRR16082819

Now it depends what is your exact goal. You may (separate approaches):

  • get mallard transcriptome as complete as possible, use kallisto just to check that a given pair of FASTQs contains bunch of reads derived from tp53
  • use STAR to align it to the genome with the contig containing TP53 gene
  • one can also try to fish out the reads out of FASTQ using tp53 cDNA sequence, but this will likely miss i.e. putative alternative 5- & 3-prime part of the gene
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    $\begingroup$ Thank you for this insightful response! For the last part, could you add more details about this step of alignment against the genome? Namely, how exactly can I confirm the contig containing the TP53 gene is present and identified using STAR? $\endgroup$
    – user18959
    Commented Feb 17 at 15:06
  • $\begingroup$ It is this contig NW_024009712 you will have to get both the genome fasta ans GTF annotation from ncbi.nlm.nih.gov/datasets/genome/GCF_015476345.1 $\endgroup$
    – darked89
    Commented Feb 17 at 16:55
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    $\begingroup$ That works great — thank you! Going to test this all out now $\endgroup$
    – user18959
    Commented Feb 21 at 9:58
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    $\begingroup$ It's part of the nf-core pipeline — it expects annotations in GTF format, but the above link only provides GFF files. I tried using gff3_ID_generator.py to generate IDs if the gff file does not have them for every feature, but the error persists $\endgroup$
    – user18959
    Commented Feb 21 at 10:42
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    $\begingroup$ Got it working now. These gtf files have a lot information that needs to be cleaned / corrected (e.g. missing gene IDs), but it appears to now run! $\endgroup$
    – user18959
    Commented Mar 4 at 5:55

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