Episode 82: Bioinformatics moments before the millennium

Mark Pallen’s bioinformatics before the millennium

Professor Mark Pallen joins Lee Katz, Andrew Page and Nabil-Fareed Alikhan to trace his move from medical microbiology to bioinformatics. His recollections span a 1977 entrance exam, sequence searches in the late 1980s and early access to bacterial genome data in the 1990s. The discussion shows how computational comparisons could guide experiments long before automated annotation pipelines.

Listen to Episode 82: Bioinformatics moments before the millennium on SoundCloud

In this episode

Assembling proteins before bioinformatics had a name

Pallen dates his first bioinformatics experience to a Cambridge entrance examination in 1977, the year Fred Sanger described his sequencing method. Given peptide sequences, he had to assemble them into a larger protein sequence by hand. He remembers looking for overlaps and joining matching fragments, much like a jigsaw. Protein sequences made this simpler than DNA assembly because there was no reverse-complement problem to consider.

He connects that aptitude with an interest in languages, including Latin, French and Esperanto. Later collaborations extended sequence-analysis thinking to texts: comparing editions of Darwin’s On the Origin of Species for additions, deletions and edits, and discussing alignment and textual changes with a theologian studying Gospel of Luke manuscripts.

An Apple Mac, urease and a decisive sequence match

At Barts Hospital, Pallen joined a molecular biology group that included Brendan Wren and Chris Clayton. Clayton had a possible Helicobacter pylori urease clone, but its weak antibody signal and lack of urease activity left its identity uncertain. The pair digitised sequencing results by having Clayton read bases from autoradiographs while Pallen entered them into an Apple Mac.

On 15 September 1989, Pallen compared the translated sequences with jack bean urease. He recalls using FASTA, or possibly fastp, with the PIR protein database—not BLAST, which came later. Around 50% amino acid identity convinced him that Clayton had cloned the urease genes. Matches switching between reading frames also helped them locate base-counting errors and correct the sequence. The work appeared in Nucleic Acids Research after rejection by Nature.

Other discoveries included a group II intron containing a reverse transcriptase and Clostridium difficile genes homologous to a butanol-fermentation pathway in Clostridium acetobutylicum. Pallen links the latter pathway to Chaim Weizmann’s First World War fermentation work and the Balfour Declaration.

A PCR typing opportunity that slipped away

Pallen recalls proposing bacterial typing with arbitrary PCR primers and a low annealing temperature. Experiments produced different banding patterns from different E. coli strains, suggesting a useful typing method. However, a journal declined the manuscript, and inconsistent profiles in subsequent experiments led the researchers to put the work aside.

About 18 months later, he saw a paper describing the approach now called RAPD. Looking back, he suspects carry-over amplicon contamination could have explained their extra bands, rather than treating that explanation as established. He keeps an image of one of the gels as a reminder about missed opportunities. His MD research also used PCR to detect the diphtheria toxin gene.

A PhD, medical internet guides and University Challenge

Gordon Dougan encouraged Pallen to undertake a PhD through a Wellcome Trust scheme for medical microbiologists. Moving from consultant status to being seen as a laboratory novice meant three years of hard graft, but it strengthened both his molecular biology and computational skills. Pallen remembers Dougan using the term bioinformatics around 1995–1996; previously, he had called his work sequence analysis.

During the PhD, Pallen wrote three articles introducing the medical profession to the internet for the British Medical Journal. Their optimism included the observation that email lacked junk mail—something his daughter later found amusing. He also captained a winning University Challenge team. Afterwards, he recalls submitting four proposals to the Wellcome Trust without success and four to the BBSRC, three of which secured project grants.

Hearing that a bacterial genome had been sequenced

Another memorable moment came when a colleague returned from an American conference with news of Craig Venter and Ham Smith’s bacterial genome sequencing. Pallen recalls hearing about Haemophilus influenzae and a second, mycoplasma genome.

For him, the breakthrough was methodological as well as biological: shotgun sequencing replaced a laborious, map-led workflow with many small fragments and computational assembly. The bioinformatics did the heavy lifting. He describes the Sanger adopting this approach and his subsequent involvement in the Campylobacter jejuni genome project.

Nick Loman and early access to the C. jejuni genome

Work on internet columns for the student BMJ brought Pallen into contact with Nick Loman, then still at school. They ran an internet workshop for medics, discovering that some participants needed help even with using a mouse. Pallen subsequently recruited Loman for a gap year at the age of 18.

The Sanger was releasing C. jejuni shotgun reads, but its workflow postponed biological interpretation until sequencing, assembly and annotation were complete. Pallen asked Loman to use the reads, BLAST comparisons against E. coli proteins and metabolic pathways to make the emerging genome accessible to researchers. Loman built a relational database and web front end in about three days, rather than the months Pallen had anticipated.

Pallen contrasts this early access with manual annotation, in which individual protein-coding genes were assessed one at a time. Their work let the research community investigate the data before the formal process finished and contributed to the eventual Nature genome paper.

Highlights

00:01:39 — Pallen’s 1977 entrance exam: assembling peptide sequences by hand
00:14:15 — Why sequence comparisons and homopolymer errors remain familiar problems
00:14:58 — Clarifying the use of FASTA rather than BLAST in early sequence searches
00:16:19 — Finding overlaps to assemble proteins, and the connection with languages
00:17:37 — Applying sequence-comparison ideas to On the Origin of Species and gospel manuscripts
00:20:27 — Early PCR experiments and the bacterial typing approach Pallen set aside
00:27:44 — Captaining a winning University Challenge team during the PhD
00:30:25 — The Sanger’s sequencing projects and Pallen’s involvement with C. jejuni
00:31:41 — Hearing Gordon Dougan use the term bioinformatics in the mid-1990s
00:37:27 — Staged genome workflows, manual annotation and interpreting data earlier

In their own words

You just lined them up and look for the overlaps and where they overlap, you join them together until you got the whole thing.

— Mark Pallen, 00:16:19

Who is talking

Mark Pallen (guest, University of East Anglia; Quadram Institute)
Lee Katz (host)
Andrew Page (host)
Nabil-Fareed Alikhan (host)

Also mentioned: Fred Sanger, Brendan Wren, Chris Clayton, Gordon Dougan, Nick Loman, Chaim Weizmann, Craig Venter, Ham Smith.

Tools and resources mentioned

FASTA, fastp, BLAST, PIR protein database, PCR, RAPD, Western blotting, Shotgun sequencing, Perl, BBC BASIC, BBC Micro, Apple Mac.

Questions this episode answers

How did Mark Pallen identify the H. pylori urease genes?

He compared translated sequences from Chris Clayton’s candidate clone with jack bean urease and found around 50% amino acid identity. Matches moving between reading frames then helped them identify and correct errors in the sequence readings.

Did Pallen use BLAST for his 1989 urease analysis?

No. After a host points out the chronology, Pallen clarifies that he recalls using FASTA, with an earlier mention of fastp as another possibility, to search the PIR protein database.

What did Nick Loman contribute to the first C. jejuni genome project?

As an 18-year-old gap-year recruit, Loman built a relational database and web front end for examining the emerging genome data. Pallen recalls that it took about three days and helped researchers investigate genes and pathways before the Sanger’s full annotation workflow was complete.

Why did Pallen stop developing his early PCR typing method?

The researchers encountered inconsistent banding patterns after an initial journal rejection and decided not to continue. Pallen later suspected contamination by previous PCR products, and recalls seeing the approach published by others about 18 months later as the method now called RAPD.

This page was written from a machine transcript of the episode and checked against it. Listen to the episode for the whole conversation.

Show notes

In this episode we talk to Professor Mark Pallen, who discusses the highlights from his long career as a medical microbiologist turned bioinformatician.

His bioinformatics journey began in 1977, the year Fred Sanger invented DNA sequencing-as-we know-it, when Mark was tasked with assembling some amino acid sequences under exam conditions. Mark explains how little was know about sequences at the time. Luckily he managed to gain a grasp of molecular biology and joined a group in the late 1908s at Barts Hospital in London, where he met Brendan Wren. Mark’s first eureka moment followed shortly afterwards, when he analysed sequences encoding the key enzyme urease from Helicobacter pylori. He also got very excited when he analysed genes from a clostridial butanol fermentation pathway, which he explains, played a central role in the formation of the state of Israel. His next big break came when he got the chance to do a PhD under Gordon Dougan. During this time, Mark not only improved his lab and bioinformatics skills, but captained a winning team in University Challenge and introduced the medical profession to the Internet. He recalls with excitement the moment when he first heard the news that a bacterial genome had been sequenced. Shortly afterwards he recruited an 18-year old gap year student, Nick Loman, to come and work with him analysing the very first Campylobacter jejuni genome. We close this episode just as the new millennium begins, with much more excitement to follow in the next episode.

Relevant links:

Butanol - https://academic.oup.com/femsle/article/124/1/61/486499
Tree-like thinking for genes, languages and gospel manuscripts - https://www.youtube.com/watch?v=8Ykj5wQs7vU

Selective bibliography

Konings, F, Perkins, MD, Kuhn, JH, Pallen, MJ, Alm, EJ, Archer, BN, Barakat, A, Bedford, T, Bhiman, JN, Caly, L, Carter, LL, Cullinane, A, de Oliveira, T, Druce, J, El Masry, I, Evans, R, Gao, GF, Gorbalenya, AE, Hamblion, E, Herring, BL, Hodcroft, E, Holmes, EC, Kakkar, M, Khare, S, Koopmans, MPG, Korber, B, Leite, J, MacCannell, D, Marklewitz, M, Maurer-Stroh, S, Rico, JAM, Munster, VJ, Neher, R, Munnink, BO, Pavlin, BI, Peiris, M, Poon, L, Pybus, O, Rambaut, A, Resende, P, Subissi, L, Thiel, V, Tong, S, van der Werf, S, von Gottberg, A, Ziebuhr, J, Van Kerkhove, MD (2021) SARS-CoV-2 Variants of Interest and Concern naming scheme conducive for global discourse Nature Microbiology 6 (7) 821 823
Pallen, MJ, Alikhan, NF (2021) Naming the Unnamed: Over 45,000 Candidatus Names for Unnamed Archaea and Bacteria in the Genome Taxonomy Database Preprints https://doi.org/10.20944/preprints2021110557v1
Pallen MJ (2021) Bacterial nomenclature in the era of genomics New Microbes New Infect 44 100942 https://doi.org/10.1016/jnmni2021100942
Pallen, MJ, Telatin, A, Oren, A (2021) The Next Million Names for Archaea and Bacteria Trends in Microbiology 29 (4) 289 298 https://doi.org/10.1016/jtim202010009
Gilroy, R, Ravi, A, Getino, M, Pursley, I, Horton, DL, Alikhan, N-F, Baker, D, Gharbi, K, Hall, N, Watson, M, Adriaenssens, EM, Foster-Nyarko, E, Jarju, S, Secka, A, Antonio, M, Oren, A, Chaudhuri, RR, Ragione, RL, Hildebrand, F, Pallen, MJ (2021) Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture PeerJ 9 e10941 https://doi.org/10.7717/peerj10941
Ravi, A, Halstead, FD, Bamford, A, Casey, A, Thomson, NM, Van Schaik, W, Snelson, C, Goulden, R, Foster-Nyarko, E, Savva, GM, Whitehouse, T, Pallen, MJ, Oppenheim, BA (2019) Loss of microbial diversity and pathogen domination of the gut microbiota in critically ill patients Microbial Genomics 5 (9) 293 https://doi.org/10.1099/mgen0000293
Connor, TR, Loman, NJ, Thompson, S, Smith, A, Southgate, J, Poplawski, R, Bull, MJ, Richardson, E, Ismail, M, Thompson, SE, Kitchen, C, Guest, M, Bakke, M, Sheppard, SK, Pallen, MJ (2016) CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online resource for the medical microbiology community Microbial genomics 2 (9) e000086 https://doi.org/10.1099/mgen0000086
Loman NJ, Pallen MJ (2015) Twenty years of bacterial genome sequencing. Nat Rev Microbiol 13:787–794. https://doi.org/10.1038/nrmicro3565
Smith O, Momber G, Bates R, Garwood P, Fitch S, Pallen M*, Gaffney V, Allaby RG (2015) Sedimentary DNA from a submerged site reveals wheat in the British Isles 8000 years ago. Science 347:998–1001. https://doi.org/10.1126/science.1261278
Kay GL, Sergeant MJ, Zhou Z, Chan JZ, Millard A, Quick J, Szikossy I, Pap I, Spigelman M, Loman NJ, Achtman M, Donoghue HD, Pallen MJ (2015) Eighteenth-century genomes show that mixed infections were common at time of peak tuberculosis in Europe. Nat
Halachev MR, Chan JZ, Constantinidou CI, Cumley N, Bradley C, Smith-Banks M, Oppenheim B, Pallen MJ (2014) Genomic epidemiology of a protracted hospital outbreak caused by multidrug-resistant Acinetobacter baumannii in Birmingham, England. Genome Med 6:70. https://doi.org/10.1186/s13073-014-0070-x
Sergeant MJ, Constantinidou C, Cogan TA, Bedford MR, Penn CW, Pallen MJ (2014) Extensive microbial and functional diversity within the chicken cecal microbiome. PLoS One 9:e91941. https://doi.org/10.1371/journal.pone.0091941
Pallen MJ (2014) Diagnostic metagenomics: potential applications to bacterial, viral and parasitic infections. Parasitology 141:1856–1862. https://doi.org/10.1017/S0031182014000134 Citations: 103
Doughty EL, Sergeant MJ, Adetifa I, Antonio M, Pallen MJ (2014) Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer. PeerJ 2:e585. https://doi.org/10.7717/peerj.585
Chan JZ, Sergeant MJ, Lee OY, Minnikin DE, Besra GS, Pap I, Spigelman M, Donoghue HD, Pallen MJ (2013) Metagenomic analysis of tuberculosis in a mummy. N Engl J Med 369(3):289–290. https://doi.org/10.1056/NEJMc1302295
Robinson ER, Walker TM, Pallen MJ (2013) Genomics and outbreak investigation: from sequence to consequence. Genome Med 5:36. https://doi.org/10.1186/gm440
Loman NJ, Constantinidou C, Christner M, Rohde H, Chan JZ, Quick J, Weir JC, Quince C, Smith GP, Betley JR, Aepfelbacher M, Pallen MJ (2013) A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4. JAMA 309:1502–1510. https://doi.org/10.1001/jama.2013.3231
Loman NJ, Misra RV, Dallman TJ, Constantinidou C, Gharbia SE, Wain J, Pallen MJ (2012) Performance comparison of benchtop high-throughput sequencing platforms. Nat Biotechnol 30:434–439. https://doi.org/10.1038/nbt.2198
Loman NJ, Constantinidou C, Chan JZ, Halachev M, Sergeant M, Penn CW, Robinson ER, Pallen MJ (2012) High-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity. Nat Rev Microbiol 10:599–606. https://doi.org/10.1038/nrmicro2850
Chan JZ, Pallen MJ, Oppenheim B, Constantinidou C (2012) Genome sequencing in clinical microbiology. Nat Biotechnol 30:1068–1071. https://doi.org/10.1038/nbt.2410
Chan JZ, Halachev MR, Loman NJ, Constantinidou C, Pallen MJ (2012) Defining bacterial species in the genomic era: insights from the genus Acinetobacter. BMC Microbiol 12:302. https://doi.org/10.1186/1471-2180-12-302
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Lewis T, Loman NJ, Bingle L, Jumaa P, Weinstock GM, Mortiboy D, Pallen MJ (2010) High-throughput whole-genome sequencing to dissect the epidemiology of Acinetobacter baumannii isolates from a hospital outbreak. J Hosp Infect 75:37–41. https://doi.org/10.1016/j.jhin.2010.01.012
Pallen MJ (2010) High-throughput sequencing as a tool in the discovery, diagnosis and epidemiology of nosocomial pathogens. Journal of Hospital Infection 76:S1. https://doi.org/10.1016/S0195-6701(10)00376-2
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Pallen MJ, Nelson KE, Preston GM (2007) Bacterial Pathogenomics (ASM Press, Washington, DC, [Edited Book]
Pallen MJ (2007) Introduction to Pathogenomics in Bacterial Pathogenomics, eds Pallen
Tobe T, Beatson SA, Taniguchi H, Abe H, Bailey CM, Fivian A, Younis R, Matthews S, Marches O, Frankel G, Hayashi T, Pallen MJ (2006) An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination. Proc Natl Acad Sci U S A 103:14941–14946. https://doi.org/10.1073/pnas.0604891103
Pallen MJ, Beatson SA, Bailey CM (2005) Bioinformatics, genomics and evolution of non-flagellar type-III secretion systems: a Darwinian perspective. FEMS Microbiol Rev 29:201–229. https://doi.org/10.1016/j.femsre.2005.01.001
Pallen MJ, Beatson SA, Bailey CM (2005) Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion. BMC Microbiol 5:9. https://doi.org/10.1186/1471-2180-5-9
Pallen MJ, Penn CW, Chaudhuri RR (2005) Bacterial flagellar diversity in the post-genomic era. Trends in Microbiology 13:143–149. https://doi.org/10.1016/j.tim.2005.02.008
Ideses D, Gophna U, Paitan Y, Chaudhuri RR, Pallen MJ, Ron EZ (2005) A degenerate type III secretion system from septicemic Escherichia coli contributes to pathogenesis. J Bacteriol 187:8164–8171. https://doi.org/10.1128/JB.187.23.8164-8171.2005
Ren CP, Chaudhuri RR, Fivian A, Bailey CM, Antonio M, Barnes WM, Pallen MJ (2004) The ETT2 gene cluster, encoding a second type III secretion system from Escherichia coli, is present in the majority of strains but has undergone widespread mutational attrition. J Bacteriol 186:3547–3560. https://doi.org/10.1128/JB.186.11.3547-3560.2004
Zhang L, Chaudhuri RR, Constantinidou C, Hobman JL, Patel MD, Jones AC, Sarti D, Roe AJ, Vlisidou I, Shaw RK, Falciani F, Stevens MP, Gally DL, Knutton S, Frankel G, Penn CW, Pallen MJ (2004) Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157:H7. Infect Immun 72:7282–7293. https://doi.org/10.1128/IAI.72.12.7282-7293.2004
Chaudhuri RR, Khan AM, Pallen MJ (2004) coliBASE: an online database for Escherichia coli, Shigella and Salmonella comparative genomics. Nucleic Acids Res 32:D296–9. https://doi.org/10.1093/nar/gkh031
Cerdeno-Tarraga AM, Efstratiou A, Dover LG, Holden MT, Pallen M, Bentley SD, Besra GS, Churcher C, James KD, De Zoysa A, Chillingworth T, Cronin A, Dowd L, Feltwell T, Hamlin N, Holroyd S, Jagels K, Moule S, Quail MA, Rabbinowitsch E, Rutherford KM, Thomson NR, Unwin L, Whitehead S, Barrell BG, Parkhill J (2003) The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129. Nucleic Acids Res 31:6516–6523. https://doi.org/10.1093/nar/gkg874
Pallen MJ, Francis MS, Futterer K (2003) Tetratricopeptide-like repeats in type-III-secretion chaperones and regulators. FEMS Microbiol Lett 223:53–60. https://doi.org/10.1016/S0378-1097(03)00344-6
Bentley SD, Maiwald M, Murphy LD, Pallen MJ, Yeats CA, Dover LG, Norbertczak HT, Besra GS, Quail MA, Harris DE, von Herbay A, Goble A, Rutter S, Squares R, Squares S, Barrell BG, Parkhill J, Relman DA (2003) Sequencing and analysis of the genome of the Whipple’s disease bacterium Tropheryma whipplei. Lancet 361:637–644. https://doi.org/10.1016/S0140-6736(03)12597-4
Roche FM, Massey R, Peacock SJ, Day NP, Visai L, Speziale P, Lam A, Pallen M, Foster TJ (2003) Characterization of novel LPXTG-containing proteins of Staphylococcus aureus identified from genome sequences. Microbiology 149:643–654. https://doi.org/10.1099/mic.0.25996-0
Pallen MJ (2002) The ESAT-6/WXG100 superfamily – and a new Gram-positive secretion system? Trends in Microbiology 10:209–212. https://doi.org/10.1016/S0966-842X(02)02345-4
Spiers A, Lamb HK, Cocklin S, Wheeler KA, Budworth J, Dodds AL, Pallen MJ, Maskell DJ, Charles IG, Hawkins AR (2002) PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide. J Biol Chem 277:39443–39449. https://doi.org/10.1074/jbc.M202790200
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Pallen M, Chaudhuri R, Khan A (2002) Bacterial FHA domains: neglected players in the phospho-threonine signalling game? Trends in Microbiology 10:556–563. https://doi.org/10.1016/S0966-842X(02)02476-9
Pallen MJ, Lam AC, Loman N (2001) Tricorn-like proteases in bacteria. Trends in Microbiology 9:518–521. https://doi.org/10.1016/S0966-842X(01)02199-0
Pallen MJ, Lam AC, Antonio M, Dunbar K (2001) An embarrassment of sortases - a richness of substrates? Trends in Microbiology 9:97–102. https://doi.org/10.1016/S0966-842X(01)01956-4
Pallen MJ, Lam AC, Loman NJ, McBride A (2001) An abundance of bacterial ADP-ribosyltransferases–implications for the origin of exotoxins and their human homologues. Trends in Microbiology 9:302–7; discussion 308. https://doi.org/10.1016/S0966-842X(01)02074-1
Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, Chillingworth T, Davies RM, Feltwell T, Holroyd S, Jagels K, Karlyshev AV, Moule S, Pallen MJ, Penn CW, Quail MA, Rajandream MA, Rutherford KM, van Vliet AH, Whitehead S, Barrell BG (2000) The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403:665–668. https://doi.org/10.1038/35001088
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Ponting CP, Pallen MJ (1999) A beta-propeller domain within TolB. Mol Microbiol 31:739–740. https://doi.org/10.1046/j.1365-2958.1999.01168.x
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Pallen M, Kiley R (1996) Medical Information on the Internet. British Medical Journal 313:1411. https://doi.org/10.1136/bmj.313.7069.1411a
Mullany P, Pallen M, Wilks M, Stephen JR, Tabaqchali S (1996) A group II intron in a conjugative transposon from the gram-positive bacterium, Clostridium difficile. Gene 174:145–150. https://doi.org/10.1016/0378-1119(96)00511-2
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Pallen M (1995) Guide to the Internet: Introducing the Internet. British Medical Journal 311:1422–1424. https://doi.org/10.1136/bmj.311.7017.1422
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Pallen MJ, Hay AJ, Puckey LH, Efstratiou A (1994) Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxin. J Clin Pathol 47:353–356. https://doi.org/10.1136/jcp.47.4.353
Mullany P, Clayton CL, Pallen MJ, Slone R, al-Saleh A, Tabaqchali S (1994) Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome. FEMS Microbiol Lett 124:61–67. https://doi.org/10.1111/j.1574-6968.1994.tb07262.x
Clayton CL, Pallen MJ, Kleanthous H, Wren BW, Tabaqchali S (1990) Nucleotide sequence of two genes from Helicobacter pylori encoding for urease subunits. Nucleic Acids Res 18:362. https://doi.org/10.1093/nar/18.2.362