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Long read alternative for FastQScreen
by Marco van Zwetselaar 14 May '24

14 May '24

Dear all, In KCRI's Illumina Basecall workflow, I always used to run FastQScreen (with GCHR38 for human, and UniVec_Core for contaminants) to detect contamination. It's slow but it gives easily interpretable output (including graphs and MultiQC integration), and if desired you very easily make it "bucket" the reads into the database(s) that they map on. When I copied the job into the Nanopore basecall workflow I was in for a surprise. The jobs took forever or ran out of requested HPC memory (if I remember well, even 96G didn't cut it for some runs). Clearly, like FastQC, FastQScreen is an oldie, and especially read mapping (which is effectively what it does) has since been optimised a lot. My intuition would be that e.g. KMA would do this in a fraction of the time. (cc-ing Philip in case he's not on this list) The only thing that FastQScreen effectively adds to the mapping is the user-friendly table and graph. Anyone keen to add a newer mapper to FastQScreen (it now has BWA and Bowtie2 as options, if I remember well)? Alternatively, suggestions for an alternative to FastQScreen? During the CoP meeting, more suggestions were made: - Use Kraken (with the added advantage of getting much more information than the "yes/no contamination" from FastQScreen), including quantification of cross-sample/species contamination - Why detect contamination on reads, when it's much easier to do this on the assembly, contaminants will come "falling out" anyway - Also: for assemblies there are tools such as CheckM (for quantifying both completeness and contamination, including within-species) Marco

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Tip: FastQC crashing on large input files
by Marco van Zwetselaar 13 May '24

13 May '24

Dear all, On the topic of FastQC: if you see FastQC failing on large fastq files, here's the fix. The issue is that FastQC crashes as it runs out of memory, but then exits with a zero exit code, and without logging an error. The reason why it runs out of memory is that its startup script sets the JVM max memory parameter (-Xmx) to 250MB. That number stems straight from the dark ages, when 2GB was a lot already. :-) Two fixes: 1. Edit the fastqc startup script, look for '-Xmx250m' and change this to e.g. '-Xmx8g'. Downside: you must remember to redo this after reinstallation or upgrade. 2. Run fastqc with '-t 32' (or some other large number). In theory this makes it multithread on 32 threads, but in practice it still runs on a single CPU[1]. The upside (despite the lack of speed benefits) is that the startup script now allocates 32 x 250MB memory. Cheers Marco [1] Presumably it uses Java (software) threads, rather than system processes / threads.

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Long read alternative for FastQC?
by Marco van Zwetselaar 13 May '24

13 May '24

Dear all, FastQC has long been the standard choice for screening Illumina reads / runs. What do you use instead for Nanopore reads? In my experience, it's always worthwhile going over the PDF report produced by MinKNOW after a run. Just like the SAV on Illumina, it gives pretty deep insight, esp. when things have gone wrong. :) But what to use if you only have the reads? nanoQC[1] was suggested in the meeting. From the MultiQC docs it appears that it can integrate Nanoplot output[2] (from the maker of NanoQC), which suggests NanoQC integrates in MultiQC as well. (I like MultiQC as it makes it easy to bundle QC outputs for the "customer".) On a separate note: is there a conventional Q cutoff for reporting ONT read quality? I picked "percentage Q17+ bases" as my analogue for the typical "pct Q30+" on Illumina, because Q30 made little sense (esp. in the early Nanopore days), and Q17 corresponds to ~98% accuracy, a decent target for MinION reads. I've always wondered if there isn't a generally agreed Q-cutoff value? Marco [1] https://github.com/wdecoster/nanoQC [2] https://github.com/MultiQC/MultiQC/issues/1074 [3] https://github.com/wdecoster/NanoPlot

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Minutes of CoP Bioinformatics meeting
by Marco van Zwetselaar 13 May '24

13 May '24

Dear all, Greetings from a mostly disconnected East Africa[*]! Thanks all who attended the CoP Bioinformatics meeting last week. I think a lot of interesting points were brought up. Rather than distributing a document with minutes, let me send out a string of emails (one per topic, for easier follow-up) with open ends that were brought up. Do chime in if you have additions or answers! Ahead of this, here's my recollection of activities mentioned in the "stand-up round", i.e. things that people are working on / would like to learn more about / will be working on: - Streptococcus pneumoniae pipeline - Klebsiella pneumoniae, hypermucous - Hybrid Illumina / Nanopore assembly - Novel sequence types found in clinical isolates - New resistance genes - Vaccination targets - Standardised cut-offs for detection - QA, assembly quality analysis - Repeated sequences detection - Evolutionary biogenetics - Microbial metagenomics Kind regards, Marco [*] Apparently there was a break in the deep sea connection to South-Africa yesterday morning, and most East African internet traffic is now being redirected through Madagascar - at crawling speed. Good to see that old fashioned email still mostly makes it through! :-)

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ONT pipeline question (again)
by Christa Twyford Gibson 13 May '24

13 May '24

Hi Bioinformatics Community of Practice Members, Now that we have more members on this listserv, I thought I’d ask this question one more time. 😊 SeqAfrica wants to set up an expert elicitation to talk about harmonization and standardization of tools for MinION data analysis and we need your input! Please respond with the answer to this question: If you conduct viral and/or microbial sequencing using an Oxford Nanopore Technologies sequencing platform, what pipeline do you use for analysis? We will invite the developers of the most used pipelines to speak at an upcoming workshop! Thank you for your input! Christa [cid:image001.png@01DAA51C.4620C130] Christa Twyford Gibson Project Manager for SeqAfrica Project SEQAFRICA (antimicrobialresistance.dk)<https://antimicrobialresistance.dk/seqafrica.aspx> chrgi(a)dtu.dk<mailto:chrgi@dtu.dk> +45 935 106 61 National Food Institute Research Group for Global Capacity Building Building 204 Henrik Dams Allé 2800 Kgs. Lyngby www.food.dtu.dk<http://www.food.dtu.dk/>

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Many purported pseudogenes in bacterial genomes are bona fide genes
by Marco van Zwetselaar 29 Apr '24

29 Apr '24

Dear all, I found this an interesting read: Cooley, N.P., Wright, E.S. Many purported pseudogenes in bacterial genomes are bona fide genes. BMC Genomics 25, 365 (2024). https://doi.org/10.1186/s12864-024-10137-0 https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-024-10137-0 It's actually more about assembly quality (and its factors) than about pseudogenes as such. I like their idea that pseudogene count could be used as an indicator of genome quality. Makes sense if you consider that it is biologically improbable to find a pseudogene where the closest reference has a functional gene. Marco

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ONT pipeline tools
by Christa Twyford Gibson 18 Apr '24

18 Apr '24

Hi Bioinformatics Community of Practice Members, SeqAfrica wants to set up an expert elicitation to talk about harmonization and standardization of tools for MinION data analysis and we need your input! Please respond with the answer to this question: If you conduct viral and/or microbial sequencing using an Oxford Nanopore Technologies sequencing platform, what pipeline do you use for analysis? We will invite the developers of the most used pipelines to speak at an upcoming workshop! Thank you for your input! Christa [cid:image001.png@01DA917D.D7B2F4E0] Christa Twyford Gibson Project Manager for SeqAfrica Project SEQAFRICA (antimicrobialresistance.dk)<https://antimicrobialresistance.dk/seqafrica.aspx> chrgi(a)dtu.dk<mailto:chrgi@dtu.dk> +45 935 106 61 National Food Institute Research Group for Global Capacity Building Building 204 Henrik Dams Allé 2800 Kgs. Lyngby www.food.dtu.dk<http://www.food.dtu.dk/>

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Bioinfo-list finally LIVE!
by Marco van Zwetselaar 14 Apr '24

14 Apr '24

Dear all, This took a while to get right, but finally we are up and running! This mailing list was set up for the SeqAfrica Bioinformatics Community of Practice, but is open to all bioinformaticians with an interest in microbial (AMR) genomics. The bioinfo-list is publicly archived at [1] (where you are probably reading this). To subscribe, send an email to bioinfo-list-join(a)seqshare.org or visit [2]. Kind regards, Marco [1] https://lists.seqshare.org/archives/list/bioinfo-list@seqshare.org/ [2] https://lists.seqshare.org/mailman/lists/bioinfo-list.seqshare.org/

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