Great results require great libraries.  Let our experienced scientists help you get the most out of your important samples.

To get the best sequencing results requires high quality input libraries. RTLGenomics is experienced in a variety of library preparation techniques. Good libraries do require quality input material. Some library preparations are more forgiving than others. For example, the quality of the DNA for amplicon library prep only has to be good enough for amplification.

For this reason, fragmented, low concentration, and even slightly inhibited samples can yield appropriate library preparations. This is in contrast to PacBio libraries which require High Molecular Weight (HMW) gDNA or RNA at high concentrations (enough to equal 10-20ug of DNA) in order to yield appropriately sized fragments during sequencing. A brief discussion of some of the preparations we use can be found under the links below.

Short Fragment Libraries

Short Fragment libraries are primarily used for Illumina and Ion Torrent sequencing.  Occasionally a short fragment library will be used on PacBio but this is not typical.  RTLGenomics offers a variety of options for short fragment library prep.  

  • Our standard approach is to use the Illumina recommended two-step protocol for generating libraries. We are also experienced with a one-step approach that uses the gene region primers as the sequencing primer during sequencing. There are positives and negatives to both approaches. Using a one-step approach allows for slightly longer amplicons because no sequence flows are allocated to the primer, however, unless the entire run is the same amplicon, different sequencing primers can interfere with each other and show a reduction in quality. On the other hand, a two-step approach increases the overall read quality of multi primer runs. However, it reduces the fragment size that can be sequenced and stitched together in paired-end sequencing. As there are two PCR steps, there is a higher chance of introducing contamination and bias. We have a number of assays available, a list is found here.

  • We have used Illumina Nextera kits, TruSeq kits and NEB Ultra kits in the past and are willing to use these in the future. Currently, our default kit is the KAPA HyperPlus kit. This is a robust kit, capable of using 6bp single or 8bp dual indexes. It allows for manual or enzymatic fragmentation and is flexible on the amount of starting material.

  • As with DNA libraries, there are a number of options for RNA libraries. The standard kit we use is the KAPA Stranded Library kit, with or without RiboErase. This kit (if samples are from certain sample types) allow for some flexibility with the quality of the RNA extract.

 

Long Fragment Libraries

Long Fragment libraries are generally 1kb or larger, however at RTLGenomics we primarily deal with 10-30kb (for PacBio) or 100kb-1mb fragments (for BioNano).  These library types are much less forgiving than short library preparations and require the highest quality starting material.

  • SMRTbell libraries generally start at 10kb and go up in size from there. For effective libraries, fragment sizes must be 5-10kb larger than the desired length of the read. This requires a number of size-selection steps, which require repair steps. Because there are numerous washes and clean-ups, a significant amount of DNA is lost by the end of the process. For these libraries to be effective, we need to start with 10-20ug of DNA. For some organisms, this amount of DNA may not be realistic, but we can suggest other options (Whole Genome Amplification for example) that are not ideal, but may be a good compromise.

  • Optical Mapping is a much different approach than sequencing. For this approach very long (>200kb) fragments of DNA are enzymatically nicked, and the nicked sites are labeled. This requires a very time-intensive extraction and nicking/labeling process. High quality DNA is required, and so we would prefer to do the extractions here. If not, submitted DNA will need to meet very strict quality metrics.