Library preparation may be done manually or on our Takara Bio Apollo 324 Robot or our Eppendorf epMotion 5075 robot depending on the type of prep and number of samples.  Some libraries are priced per sample, others have a setup fee with an additional cost per sample. The robotic preps are limited to the number of libraries that are done at the same time and are priced accordingly (ie. 6, 8, 96) Please contact Mark for more details.

For All Preps

Quality: A better quality sample will give a better library. Nucleic acids should have absorbance ratio values A260/A280 of 1.8–2.0. Longer length genomic DNA is better and for some preps necessary. RNA quality should be checked with the bioanalyzer before library preps and is not included in the fee. RINs of 8-10 are best for most preps. If the RIN is less, talk to us about possible preps.

Quantity: See chart below for specifics for prep type. We appreciate more DNA or RNA if you have sufficient material for 2X or more, especially if you have determined concentration by a spectrophotometer such as nanodrop rather than a fluorometric method such as Qubit.  We will double check the concentration with the Qubit fluorometer before starting. You must supply enough extra DNA or RNA beyond the required amount for the prep.  An additional 2-5 mL is recommended. This is included in the library prep fee.

PCR Cycles: We will use a recommended default value for the number of PCR cycles in the protocol’s amplification step.  Notify us if you would like to change this number.

Size Determination: After library prep is complete, the size distribution must be checked with the bioanalyzer HS-DNA chip or TapeStation HS DNA tape.  This is not included in the prep fee.

Size Selection: Some preps will need additional size selection.  We will notify you if that is the case.

Library Quantification: We recommend the library prep concentration to be determined by qPCR before sequencing for many of our preps.  This is an extra fee.

Pooling: Library pooling is included in the library prep fees.  Let us know how you would like your libraries pooled and in what ratios before we start the prep.  Selection of barcodes for the pool is important to plan before library prep is started.

Library Prep Minimum concentration (ng/mL)* Minimum Volume for prep and QC (mL) ! Quantity DNA or RNA needed for prep (ng) Insert or PCR amplicon size bp (w/o adapters)
  DNA
PrepX DNA 1.5 105 150 220, 320, 520, 870
TruSeq DNA Nano 350 bp or 550 bp insert 1.5 or 2.5 105 150 or 250 350, 550
TruSeq DNA PCR-Free 350 bp or 550 bp insert 105 or 205 105 1050 or 2050 350, 550
Nextera XT DNA 0.2 7 1 200-2000 (835)
Illumina Nextera Flex DNA - Large Genome 3.5-17 35 100-500 300-350
Illumina Nextera Flex DNA - Small Genome 0.05-17 35 1-500 300-350
Nextera Mate Pair Gel-Free 14 80 1000 2-15kb (2.5-4kb)
Nextera Mate Pair Gel-Plus 13 312 4000 Gel size selection
TruSeq ChIP 0.17 65 10 150-200 (custom)
  RNA
PrepX Robotic PolyA + Stranded RNA 20 55 1000 100-200
PrepX Robotic RiboZero + Stranded RNA 3.6-180 32 1000-5000 100-200
Lexogen QuantSeq 3' mRNA-Seq FWD 2-400 8 10-2000 (500) 100-200
TruSeq Small RNA  200 7 1000 22-30
16s amplicon PCR library 2.5 7 12.5 460
  CUSTOM
Ask us about methylation, Hi-C, other preps of interest

*It is appreciated if you can supply a higher concentration and larger volume than the minimum recommendation. If you have read your concentration with a spectrophotometric method instead of a fluorometric method, you may need to supply much more than the minimum concentration to have enough for a prep.

! You must supply enough extra DNA or RNA beyond the required amount for the prep for QC purposes.  An additional 2-5 mL is recommended.

Library specific information

PrepX DNA

  • Whole Genome Sequencing library. Similar to the TruSeq Nano preps. Prep done on our Takara Bio Apollo 324 robot.

TruSeq DNA Nano 350 bp or 550 bp insert

  • Whole Genome Sequencing library. Manual prep with mechanical fragmentation and adapter ligation. AMPure XP magnetic beads are used for the size selection.

TruSeq DNA PCR-Free 350 bp or 550 bp insert

  • Whole Genome Sequencing library. Manual prep with mechanical fragmentation and adapter ligation but no PCR amplification step. Removes PCR bias.  More starting material is required.

Nextera XT DNA

  • Whole Genome Sequencing library. A manual prep for small genomes <20Mb (microbial genomes, amplicons, plasmids, etc)
  • 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.
  • It is difficult to get even number of reads when pooling Nextera XT libraries due to the wide size distribution.

Illumina Nextera Flex DNA

  • Whole Genome Sequencing library that uses Bead-linked transposome. Results similar to TruSeq Nano DNA.
  • Supports large complex genomes, small genomes, plasmids, amplicons, gram positive/gram negative bacteria, fungi, or a range of plant and animal species
  • At some DNA input levels, accurate quantification of DNA either before or after library prep can be omitted. Small genome and large genome require different input amounts.
  • Robotic library prep single samples up to 16 samples or in sets of 8 above 16 samples. Setup fee for prep plus per sample fee.

Nextera Mate Pair

  • There are 2 options for manually prepped DNA mate pair libraries.
    • 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.

TruSeq ChIP

  • Manual prep using Illumina kit for Chromatin immunoprecipitation sequencing (ChIP-Seq)

PrepX Robotic PolyA + Stranded RNA

  • This process starts with total RNA and involves the purification of the poly-A containing mRNA, conversion to cDNA, and finally the ligation of Illumina adapters.  Done on the Apollo 324 robot.
  • Submission in sets of 6 gets discount.
  • If the starting material is mRNA, please contact Mark for more details.

PrepX Robotic RiboZero + Stranded RNA

  • Protocol starts with total RNA, removes ribosomal RNA then converts mRNA to cDNA and ligation of Illumina adapters.  Done on the Apollo 324 robot.
  • Submission in sets of 6 gets discount.

Lexogen QuantSeq 3' mRNA-Seq FWD

  • QuantSeq protocol generates only one fragment per transcript starting with total RNA resulting in extremely accurate gene expression values and the sequences obtained are close to the 3’ end of the transcripts. Lower quality RNA could be used since 3’ end of transcript is targeted in this protocol. An alternate protocol is available for lower starting amounts of RNA. If you are interested in full transcription another protocol should be used such as the PrepX Robotic PolyA + Stranded RNA.
  • Robotic library prep in sets of 8. Setup fee for prep plus per sample fee.