4. Illumina Sequencing

Which statements are correct with respect to Illumina’s cluster generation process?

1) Oligonucleotides complementary to the P5 and P7 adapters are located on the flow cell

2) No polymerase is needed for bridge amplification

3) If one overestiamtes the concentration of an Illumina library this leads to too few clusters on the flowcell

4) If two different clusters overlap too much, they will generate no useable sequencing data

5) If cluster generation would be 100% efficient one would generate over 1000 DNA molecules from one starting DNA molecule after 10 cycles

Which statements regarding the Illumina sequencing technology are correct?

1) As all DNA molecules in one cluster are identical, the same fluorescent nucleotide gets incorporated in (almost) all those molecules during one cycle

2) Illumina sequencing requires detection of single fluorescent molecules

3) The fluorescent label is attached to the triphosphate of the nucelotides

4) For sequencing, the four different fluorescently labeled nucleotides are added one after the other to the flow cell

5) The lower the efficiency of the nucleotide incorporation during the sequencing by synthesis reaction, the faster drops the quality score over the read length

Which statements regarding Illuminas sequencing platforms are correct?

1) Up to 5 million reads are generated on a flow cell per sequencing run on the latest machine (Nova-Seq)

2) The longest read length on most machines is currently 2x800 bp

3) Patterned flow cells allow to generate more clusters on a flow cell

4) Private persons can have their genome sequenced for ~150,-€

5) 2-channel scanning is a recent improvement that increases the read length

Which statements regarding the generation of Illumina libraries for genome sequencing are correct?

1) All Illumina libraries contain P5 and P7 sequences on their ends to enable cluster amplification

2) Nextera libraries can be made without shearing DNA as the concentration of the transposase determines the insert size distribution of the libraries

3) SPRI beads are used to load the library on the flowcell

4) Indices are introduced into libraries to allow multiplexing of libraries within a sequencing lane

5) Genomic DNA needs to get sheared since Illumina sequencing does not work well with large (>1kbp) inserts

Which statement regarding the file format of sequencing data from Illumina are correct?

1) Illumina data is provided in FASTQ format

2) Illumina data are provided as image files for the user

3) The “@” indicates always the start of the sequence identifier line

4) The following characters indicate a processing error for an Illumina sequence  “’*((((***+))%%%++)(%%%%).1*”

5) A quality value of 30 is better than a quality value of 40 in Illumina data