Library preparation

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For Illumina:

ExperimentMaterialSequencing schemeinsert size,
w/o adapters [bp]
sequencing de novo,
small genomes (bacteria, etc.)
sequencing de novo,
large genomes (eucariotes, etc.)
genome resequencing10µg<DNAMP&PE>1kbp & 180-210
50ng<DNA<10µgPE 2x50bp
PE 2x75bp
DNA<50ngPE 2x50bp
PE 2x75bp
ChipSeq1ng<DNASR 50bp
PE 2x50bp
exome sequencing5µg<DNAPE 2x75bp180-210
DNA<0.5µgPE 2x50bp130-280
mRNA profiling30µg<total RNAPE 2x50bp100-150
small RNA analysis SR 35bp

  • there is no any "universal" sequencing scheme. It is always a compromise between contradictory requirements. The main factor for resolving of the compromise is a biogical task.
  • even "best planned" sequencing may require thinking and corrections during experiment. For example, suppose, that it is planned, that some particular size range will be obtained during shearing and taken during size selection. But shearing is not completely predictible. If it gives another size distribution it would be necessary to decide what to do:
» take another portion of material and repeat shearing with corrected regime
» shift size range for the selection from the gel
» take the same size range regardless to the decrease amount of material

Depending on biological task one of the dicisions is correct.

[править] Genome resequencing

  • best strategy: MP for large rearrangements, PE — for SNP's and small rearrangements
  • MP is obligatory for analysis of large genome rearrangements. The longer fragments are used for MP-library construction:
» the more starting DNA is necessary: ~50-100µg for 5kb, ~10-20µg for 1-1.5kb
» the larger virtual coverage will be reached
  • to recognize "short" InDel's PE-fragment length variation should be less, than InDel size:
» more material is necessary for preparation of library with small fragment length variation (with the same complexity)
» to reach maximal complexity it is better to select "narrow DNA size range" in the peak of length distribution of sheared DNA (~200bp)
» the smallest possible length variation which we got is ~50bp (when cutted 20-30bp range)
» if starting amount of DNA is low, but it is necessary to obtain as much as possible complexity, it is better to take "all smear" (up to ~300bp)
  • 75bp reads more convenient for analysis of small InDel's (have more chances to overlap them)

[править] ChipSeq

  • size of DNA fragments after ChIP is ~100-600bp (ultrasound). We perform additional ultrasound shearng to reach ~100-300bp:
» more fragments
» better sequence quality
  • size range: 80-260
» low limit — not to sequenci adapters
» upper limit — practically end of smear
  • for most ChIP's single 50bp read sequencing is enough:
» cheaper
» we are interested in location of ChIP fragments on genome. In most cases one 50bp read is enough for localization

[править] Enrichment

  • Agilent, Sure Select kit
  • sequencing task is similar to "genome resequencing", but restricted to exon regions.
» for >5µg of starting DNA: for InDel analysis it is preferable to keep library size variation as small as possible (180-210bp)
» for <0.5µg of starting DNA: to reach maximal complexity of the library it worth to take practically all smear (130-280bp)
  • 75bp reads more convenient for analysis of small InDel's (have more chances to overlap them)

[править] mRNA profiling

  • RNA profilign should be strand-specific: either SOLiD or dUTP-protocol
  • PE is obligatory for analysis of splicing
  • for comparison of expression profiles it is very important, that shearing and fragment selection were performed identically as mach as possible. Otherwise, errorneous systematic differences may appear
  • transcriptome is not so large as genome (~0.1% ??). 50bp reads are good enough for mapping. 75bp reads give practically no additional information, but
» more expensive
» have more seq. errors (more complex analysis)
  • library fragment sizes: compromise is 100-150bp. Reasons:
» small fragments are necessary to fish out small mRNA's
» average human exon length is ~170bp. Only those library fragments, which overlap exon/exon boundary are useful for mRNA splicing analysis. In this respect large library fragments are preferable
» the smaller library fragment length variation, the easier to perform splicing analysis
  • ribosomal RNA is ~90% of total RNA. It is necessary to reduce amount of ribosomal RNA reads somehow:
ds cDNA synthetised onreads on ribosomal RNA [%]remarks
total RNA~50%low loss of material, profiling of procariotes
one-time purified mRNA (w/o shearing)~??%better 3'-end representation
ribominus purified mRNA (w/o shearing)~??%polyA- RNA
sheared two-times purified mRNA~??%standard procedure, better 5'-end representation

[править] small RNA

  • 35bp sequencing is enough for 18-30nt small RNA's
  • normally, small RNA complexity is low, so it worth to use multiplexing

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