Illumina library prep service is charged per sample.  See HTS fees for costs.  TruSeq library prep supports up to 24 indexes (96 if dual indexing) for multiplexing.  Please note that bioanalyzer QC check is not included in the price of library prep.  At the end of 2013, the CGRB Core Facilities purchased the Wafergen Biosystems Apollo 324 NGS Library Prep System.  This allows for automated DNA, stranded RNA, ChIP, and small RNA library prep.  Wafergen sells PrepX library prep reagents which are used with the Apollo instrument.  This robotic system is not compatible with Illumina TruSeq and Nextera XT kits, but the CGRB Core Lab still offers these as a manual library prep service. 

DNA-Wafergen/TruSeq Nano/TruSeq PCR-Free

  • A260/280 ratio of DNA prior to library prep should be 1.8-2.0. 
  • We ask for the following amounts of DNA in a volume of <100uL, if possible.  Less can be used, but this is the recommended.  We will double check the concentration with the Qubit fluorometer before starting.
    Wafergen- 300ng
    Nano- 100ng for 350bp insert & 200ng for 550bp insert
    PCR-Free- 1μg for 350bp insert & 2μg for 550bp insert
  • The following approximate insert sizes are available for each prep type.  AMPure XP magnetic beads are used for the size selection.  If you need a very specific and narrow insert size, please request to have the library agarose gel size selected (extra cost).
    Wafergen- 220bp, 320bp, 520bp, & 870bp insert sizes
    Nano & PCR-Free- 350bp & 550bp insert sizes
  • Default number of cycles during PCR enrichment is the following.  Notify us if you want this changed.
    Wafergen- 10 cycles
    Nano- 8 cycles
  • After library prep is complete, the size distribution must be checked with the bioanalyzer HS-DNA chip.  This is not included in the prep fee. 

ChIP DNA- Wafergen/TruSeq ChIP

  • A260/280 ratio of DNA prior to library prep should be 1.8-2.0. 
  • We ask for 10ng of ChIP DNA in a volume of <15uL for Wafergen prep and <60uL for the TruSeq prep, if possible.  Less can be used, but this is the recommended.  We will double check the concentration with the Qubit fluorometer before starting.
  • Default insert size is 150-200bp (total fragment size w/ adapters is 250-300bp).  If you want a different size range selected, please notify us.
  • Default number of cycles during PCR enrichment is 18 cycles.  Notify us if you want this changed.
  • After library prep is complete, the size distribution must be checked with the bioanalyzer HS-DNA chip.  This is not included in the prep fee. 

Nextera XT DNA (for microbial genomes, amplicons, plasmids, etc.)

  • A260/280 ratio of DNA prior to library prep should be 1.8-2.0. 
  • We ask for 1ng of DNA in a volume of <5uL, if possible.  We will double check the concentration with the Qubit fluorometer before starting.
  • Insert sizes range from 200-2000bp with a median insert size of 835bp (total fragment size w/ adapters range from 300-2100bp).  Important: Shorter insert libraries will preferentially get sequenced on the flow cell.  Therefore, the median sequenced insert size will always be shorter than the median insert size indicated from the bioanalyzer. 
  • Default number of cycles during PCR enrichment is 12 cycles.  Notify us if you want this changed.
  • After library prep is complete, the size distribution must be checked with the bioanalyzer HS-DNA chip.  This is not included in the prep fee.
  • Important: Nextera XT libraries run on the HiSeq 2000 require a different sequencing primer kit that must be purchased for additional cost. This is not required for the MiSeq.
  • Important: Terminal 5' and 3' 50bp will have poor coverage due to the transposome technology used.  Therefore, if you need the entire sequence of a 300bp amplicon, then please be sure the amplicon is 400bp.
  • Important: It is difficult to get even number of reads when pooling Nextera XT libraries due to the wide size distribution.

Nextera Mate Pair

  • There are 2 options for DNA mate pair libaries.
    Gel-Free- Yields fragment distribution ranging from 2-15kb, with a median fragment size between 2.5-4kb.
    Gel-Plus- Fragment size and distribution determined by gel based size selection.  Can pick a narrower range of fragment sizes than the gel-free method.
  • High-quality, high molecular weight gDNA is very important for mate pair libraries.  If degraded DNA is used then the size range after tagmentation may be too short.  Agarose gel assessment should show the majority of the gDNA band >50kb.
  • We ask for the following amounts of DNA, if possible.  Less can be used, but this is the recommended.  We will double check the concentration with the Qubit fluorometer before starting.
    Gel-Free- 1ug in a volume of <76uL
    Gel-Plus- 4ug in a volume of <308uL
  • Default number of cycles during PCR enrichment is 10-15 cycles.  Notify us if you want this changed.
  • After library prep is complete, the size distribution must be checked with the bioanalyzer HS-DNA chip.  This is not included in the prep fee.

RNA- Wafergen stranded/TruSeq (non strand-specific)

  • This process involves the purification of the poly-A containing mRNA, conversion to cDNA, and finally the ligation of Illumina adapters.  Poly-A enrichment included in the cost of TruSeq prep, but not the Wafergen prep.
  • Quality of total RNA must be checked with the bioanalyzer prior to starting.  This is not included in the prep fee.
  • We ask for 1ug of total RNA in a volume of <50uL for the TruSeq prep and Wafergen polyA enrichment, if possible.  Less can be used, but this is the recommended.  We will double check the concentration with the Qubit fluorometer before starting.
  • Default insert size is 100-200bp (total fragment size w/ adapters is 220-300bp).  If you want a different size range selected, please notify us. 
  • For those doing paired end runs for de novo assembly, it is recommended to gel size select in order to have a tighter size distribution (similar to the DNA preps).  Please notify us if you need your library size selected.
  • Default number of cycles during PCR enrichment is 15 cycles.  Notify us if you want this changed.
  • After library prep is complete, the size distribution must be checked with the bioanalyzer HS-DNA chip.  This is not included in the prep fee.

If the starting material is mRNA, please contact Mark for more details.