ESPM 131 Lecture Notes - Lecture 16: Ion Semiconductor Sequencing, Rpob, Base Pair

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Lecture 16-17: Microbial Community Analysis
Complexity of soil: physical and chemical heterogeneity, complex 3D structure
Biomarkers : mols of bio origin that are used to indicate processes and indicate presence of
organisms
Should be unique to an organism → indep of env inluence
Ideal:
Found in all microbes
Taxon rank and leads to sp/subsp
Can be isolated from living microbes
Doesn’t persist “outside” microbes
Can detect in v small quant
Options:
Nuc acids/proteins (NAs meet most of criteria)
lipids/polysaccharides
Metabolites
Nucleic acids as biomarkers:
PCR increases detection (1983)
Nuc acids evolve w/ host
Exception: horiz gene transfer
struct/housekeeping genes are not/rarely transferred - more likely to
have co-evolved, commonly used are rRNA (16/18S)
Universal phylogenetic tree based on SSU rRNA - conserved/variable
regions
16S RNA : 1.5k bp, conserved and var regions
Alternate biomarkers: limited databases
gyrB : gyrase subunit, evolves faster than SSU
recA : recombinase A, evolves faster than SSU, higher resolution (more variable)
rpoB : rib polymerase, single copy/genome
ITS regions : high var, choice for fungi
Processes:
Extraction : cell lysis, DNA protection and recovery, purification
PCR : amplifies lower copy-level genes, add primers and DNApoly, heat to sep, rpt
Limitations:
Bias from universal primers
Variation in amplification eficiency
Primer annealing
Gene copy number varies (1-20), so can’t be used to quantify unless
number is known
Chimeras
Substrate exhaustion : most abundant molecules amplified first, not
enough reagents to amplify least abundant
Sequencing : cost per gigabase dec exponentially
Old: Sanger’s, Pyrosequencing, Ion torrent (pH sensing)
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Comple(cid:481)it(cid:482) o(cid:321) soil: ph(cid:482)si(cid:283)al and (cid:283)hemi(cid:283)al hete(cid:418)o(cid:322)eneit(cid:482), (cid:283)omple(cid:481) (cid:1682)d st(cid:418)u(cid:283)tu(cid:418)e. Bioma(cid:418)ke(cid:418)s : mols o(cid:321) (cid:282)io o(cid:418)i(cid:322)in that a(cid:418)e used to indi(cid:283)ate p(cid:418)o(cid:283)esses and indi(cid:283)ate p(cid:418)esen(cid:283)e o(cid:321) o(cid:418)(cid:322)anisms. Should (cid:282)e uni(cid:417)ue to an o(cid:418)(cid:322)anism indep o(cid:321) en(cid:475) in(cid:500)luen(cid:283)e. Nu(cid:283) a(cid:283)ids/p(cid:418)oteins (cid:1827)nas meet most o(cid:321) (cid:283)(cid:418)ite(cid:418)ia(cid:1828) St(cid:418)u(cid:283)t/housekeepin(cid:322) (cid:322)enes a(cid:418)e not/(cid:418)a(cid:418)el(cid:482) t(cid:418)ans(cid:321)e(cid:418)(cid:418)ed - mo(cid:418)e likel(cid:482) to ha(cid:475)e (cid:283)o-e(cid:475)ol(cid:475)ed, (cid:283)ommonl(cid:482) used a(cid:418)e (cid:418)rna (cid:1827)(cid:1680)(cid:1685)/(cid:1680)(cid:1687)s(cid:1828) Uni(cid:475)e(cid:418)sal ph(cid:482)lo(cid:322)eneti(cid:283) t(cid:418)ee (cid:282)ased on ssu (cid:418)rna - (cid:283)onse(cid:418)(cid:475)ed/(cid:475)a(cid:418)ia(cid:282)le (cid:418)e(cid:322)ions. 16s rna : (cid:1680). (cid:1684)k (cid:282)p, (cid:283)onse(cid:418)(cid:475)ed and (cid:475)a(cid:418) (cid:418)e(cid:322)ions. G(cid:482)(cid:418)b : (cid:322)(cid:482)(cid:418)ase su(cid:282)unit, e(cid:475)ol(cid:475)es (cid:321)aste(cid:418) than ssu. (cid:418)e(cid:283)a : (cid:418)e(cid:283)om(cid:282)inase a, e(cid:475)ol(cid:475)es (cid:321)aste(cid:418) than ssu, hi(cid:322)he(cid:418) (cid:418)esolution (cid:1827)mo(cid:418)e (cid:475)a(cid:418)ia(cid:282)le(cid:1828) Its (cid:418)egions : hi(cid:322)h (cid:475)a(cid:418), (cid:283)hoi(cid:283)e (cid:321)o(cid:418) (cid:321)un(cid:322)i. E(cid:481)t(cid:418)a(cid:283)tion : (cid:283)ell l(cid:482)sis, dna p(cid:418)ote(cid:283)tion and (cid:418)e(cid:283)o(cid:475)e(cid:418)(cid:482), pu(cid:418)i (cid:283)ation. Pcr : ampli es lo(cid:476)e(cid:418) (cid:283)op(cid:482)-le(cid:475)el (cid:322)enes, add p(cid:418)ime(cid:418)s and dnapol(cid:482), heat to sep, (cid:418)pt. Gene (cid:283)op(cid:482) num(cid:282)e(cid:418) (cid:475)a(cid:418)ies (cid:1827)(cid:1680)-(cid:1681)(cid:1679)(cid:1828), so (cid:283)an"t (cid:282)e used to (cid:417)uanti(cid:321)(cid:482) unless num(cid:282)e(cid:418) is kno(cid:476)n.

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