Eureka Genomics® Next Generation Sequencing Services

• After a sample of isolated nucleic acid
  • DNA
  • RNA
  • Prepared Library

 has been prepared as a library, QC analysis occurs. Libraries can be prepared by Eureka Genomics. Alternatively prepared libraries can be submitted to Eureka Genomics for sequence data generation. Libraries that pass QC undergo cluster generation for hybridization and amplification on an Illumina Flowcell. Depending on the type of library either a single end or a paired end flow cell will be used. The flowcell with clusters is used to generate sequence data on a Genome Analyzer IIx.

Cluster Generation

1. Single End Non Indexed, microRNA Non Indexed, and microRNA Indexed Libraries, can be hybridized and amplified on a Single End flowcell only.

2. Paired End Indexed and Paired End Non Indexed Libraries intended for Paired End Sequencing can be hybridized and amplified on a Paired End flowcell only.

3. Paired End Indexed and Paired End Non Indexed Libraries intended for Single End Sequencing can be hybridized and amplified on a Single End flowcell only.

Sequence Data Generation on an Illumina Genome Analyzer GAIIx

1. Single End Libraries can be used in 36, 51 or 76 cycle Single End Sequencing. Each cycle represents a single base incorporation/extension. For example 36 cycle single end sequencing will yield sequencing reads that are 36 bases long.

2. Paired End Libraries have the option of 36, 51 or 76 cycle Paired End Sequencing. The cycling occurs in both directions. For example, in a 36 cycle Paired End Sequencing, 36 bases are sequenced from one end of the library construct and 36 bases are sequenced from the other end of the library construct. This yields a total base output of 72 bases for that molecule.

3. Two types of multiplex sequencing are available for Paired End Indexed Libraries.

a. Paired End Multiplex Sequencing is available in 36, 51 or 76 cycles. Up to 12 samples can be uniquely tagged with index sequences and can be combined into a single lane of a flowcell for Multiplex Sequencing. During data analysis the individual samples in the lane can be parsed out based upon their unique index tags.

b. Single End Multiplex Sequencing is available in 36, 51 or 76 cycles. In order to accommodate index sequencing, a 36 cycle single end multiplex sequencing will yield 29 bases, 51 cycle will yield 48 bases and 76 cycles will yield 68 bases of sequence data. Up to 12 samples can be uniquely tagged with index sequences and can be combined into a single lane of a flowcell for Multiplex Sequencing.

4. mRNA Libraries:

a. Paired End Libraries generated from mRNA can be used in 36, 51 or 76 cycle Single End Sequencing. Each cycle represents a single base incorporation/extension. For example 36 cycle sequencing will yield sequencing reads that are 36 bases long.

b. Paired End Libraries generated from mRNA have the option of 36, 51 or 76 cycle Paired End Sequencing. The cycling occurs in both directions. For example, in a 36 cycle Paired End Sequencing, 36 bases are sequenced in from one end of the library construct and 36 bases are sequenced from the other end of the library construct. This yields a total base output of 72 bases for that molecule.

c. Paired End Multiplex Sequencing is available in 36, 51 or 76 cycles for Paired End Indexed Libraries generated from mRNA. Up to 12 samples can be uniquely tagged with index sequences and can be combined into a single lane of a flowcell for Multiplex Sequencing. During data analysis the individual samples in the lane can be parsed out based upon their unique index tags.

d. Single End Multiplex Sequencing is available in 36, 51 or 76 cycles for Paired End Indexed Libraries generated from mRNA. In order to accommodate index sequencing, a 36 cycle single end multiplex sequencing will yield 30 bases, 51 cycle will yield 49 bases and 76 cycles will yield 69 bases of sequence data. Up to 12 samples can be uniquely tagged with index sequences and can be combined into a single lane of a flowcell for Multiplex Sequencing.

5. MicroRNA Libraries: Two types of Single End Sequencing are available.

a. 32 cycle Single End Sequencing is available for Single End microRNA libraries.

b. 35 cycle Single End Multiplex Sequencing is available for Single End Indexed microRNA Libraries. In order to accommodate index sequencing, a 35 cycle single end multiplex sequencing yields 32 bases of sequence data. Multiple samples can be uniquely tagged with index sequences and can be combined into a single lane of a flowcell. During data analysis the individual samples in the lane can be parsed out based upon their unique index tags.