[Verse 1] Sonicate the strands, shear 'em down to size, tagmentation's quicker, enzyme cuts and ties, size selection combs the beads through the mess, short fragments wander off, long ones pass the test. PCR fires up, primers find their mark, denature, anneal, extend — cycling in the dark, polymerase stumbles, drops a wrong base in, GC-rich regions starve while AT wins, duplicates pile up from the same template ghost, chimeras splice two strangers into one host. [Chorus] Extract it, fragment it, amp it, then you read it, every artifact's a clue if you know how to read it, adapters, indexes, barcodes on parade, watch for index hopping when the pools cascade, cut it, tag it, call it — trust but verify, Sanger in the corner keeping short reads honest, why. [Verse 2] Library prep stitches adapters on the ends, UMIs are molecular tags, your truest friends, multiplex a hundred samples in one lane, but leaky index hopping mixes up the chain, hybrid capture baits your exons out the crowd, amplicon panels shout their target loud, Sanger's the old sage, slow but never lies, four-color ladder, capillary-sized, gold standard validation when your caller's unsure, run it through the Sanger, settle the score. [Chorus] Extract it, fragment it, amp it, then you read it, every artifact's a clue if you know how to read it, adapters, indexes, barcodes on parade, watch for index hopping when the pools cascade, cut it, tag it, call it — trust but verify, Sanger in the corner keeping short reads honest, why. [Verse 3] Illumina clusters glow in cycles of light, paired-end reads shake hands across the insert's flight, phasing and pre-phasing blur the tail-end call, quality decays like a whisper down the hall, patterned flow cells, two-color chemistry cheats, poly-G tails when the dark cycle repeats, PacBio circles its own read seven times, HiFi consensus scrubs away the crimes, Nanopore reads the wiggle of the ion through the pore, signal to bases, modifications and more, ultra-long strands span the repeats that hide, Hi-C folds chromatin, optical maps the wide, Strand-seq sorts homologs, bisulfite burns C to U, EM-seq spares the strand — a gentler read-through. [Bridge] FFPE deamination fakes a C-to-T scar, cfDNA fragments short as nucleosome bars, low input trembles, contamination creeps in the well, substitutions haunt Illumina, indels haunt the nanopore cell, homopolymers trip the long-read call, know your error profile or you'll misread it all, replicates, controls, batch effects tracked with care, metadata's the map — don't sequence unaware. [Verse 4] Lander-Waterman whispers the coverage you're owed, thirty-fold times one-fifty base reads down the road, divide by genome length, that's your lambda born, e to the negative lambda — the lacuna left unworn, a lacuna's a gap, a silence in the sea, solve that fraction and you'll know your blind spot's decree, three-point-one gigabases, run the numbers straight, plan your depth before you seal the plate.
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