Important Considerations    

Before submitting your order for ACGT, Inc. NGS services, please consider the following issues:

Library Construction

Library preparation can take from 2-5 days depending on the application. The input material is fragmented DNA (if the source is RNA, it is converted to cDNA). Typically, libraries consists of carefully size-selected fragments which range from 300 to 800 bp (shorter for ChIP-Seq) which are ligated with specific adapters and amplified with a PCR reaction. Once a library has been constructed and validated, cluster generation takes an additional day. The library is bound to the flow cell by hybridizing the fragments to a lawn of oligos complementary to the adapter sequences. Bridge amplification is performed to create millions of dense clusters.

Run Length

The clusters generated on cBot are sequenced by incorporation of fluorescent nucleotides (one base/cycle). The TruSeq sequencing process takes 1.5-15 days depending on read length. Typically, paired end runs of 40 to 50 cycles per end read (3-4 days) are sufficient for most types of applications, although de novo sequencing and metagenomic analysis may require longer read lengths, while ChIP-Seq reads are shorter.

Paired Ends

Sequencing from both sides of the adapter-ligated fragments can be performed. The paired end protocol essentially flips the fragments attached to the flow cell and sequences them from the other direction, after completion of the first read. Besides getting more data per read, paired end sequencing allows more positional information to be obtained from the data, facilitating the evaluation of alternate splice junctions, In/Dels, etc. The standard paired end protocol can evaluate sequences 200-700 bases apart. Clients wishing to evaluate longer distances (3-5 kb) between sequences can use the Mate-Pair protocol to generate their libraries and should contact ACGT, Inc. regarding the requirements for this service. Note: Paired end samples cannot be run on the same flow cell as single read samples.

Coverage

Because read length using Illumina technology is relatively short, multiple fold levels of sequence coverage are recommended in order to ensure efficient assembly of the contigs. For re-sequencing of genomes and transcriptomes, and ChIP analysis, we recommend a minimum of 10-fold coverage, while de novo sequencing requires at least 100-fold coverage. Polyploid genomes, such as those of many plants, require even higher coverage levels. The level of coverage in combination with the expected sequence size and the number of sequencing cycles per run determines the amount of flowcell space (number of lanes) that would be required for the experiment (see Table below)

* 7 lanes per 1 flow cell

Multiplexing

Clients wishing to run more than one sample per flowcell lane can take advantage of the Illumina indexing system. A six base sequence index is added to the adapter and sequenced by an additional short run. Twelve DNA and 48 RNA unique indexes are available. Please note that the number of readable clusters per sample will drop proportionally with the number of indexes used per lane and that the use of index sequencing still requires that a separate library be prepared for each sample.

Project Timeline

ACGT, Inc. will do its best to process samples in the order received. To use the instrument in the most efficient manner and to minimize the cost to the user, we will attempt to accumulate a full set of seven (single or multiplexed) samples before initiating a run. Clients who do not wish to wait until a full set of samples has accumulated will have the option of expediting their order by agreeing to pay the cost of the unused lanes, or to share this cost with another researcher. Please contact us if you would like to have your order expedited, or to get a cost estimate for your project. NOTE: For some applications, we do not have the necessary kits on hand and it may take 2-3 weeks to receive them from Illumina or other providers.