Yes, a 6 – 8 nt long molecular index
Yes.
A 6 – 10 nt long randomized sequence, refered to as a unique molecular index (UMI) can be introduced between the adapter sequence and target sequence of each primer. With molecular indices, PCR duplication events can be distinguished from unique priming events. Also, by using this random sequence, cluster calling can be easily accomplished on Illumina platforms. Illumina platforms rely on the initial rounds of sequencing for cluster calling and an even nucleotide sequence (25 % of A, C, G, and T) is maintained at each of these positions. If these random nucleotides are not included, be sure to design and combine your targeted primers in such a way that the first 5 nts are equally balanced within the final lane mix
Similarly, a sample barcode (a defined sequence that is unique to an individual sample) can also/instead be added, ideally during first strand synthesis. To include sample barcoding you will need individual target-specific reverse-transcription primers for each sample you wish to pool for sequencing.
Partial Illumina P7 Adapter Sequence (Read 2) for First Strand Synthesis Primer:
5' GTTCAGACGTGTGCTCTTCCGATCT – NNNNNN(NN) – Target sequence (= cDNA sequence) 3'
NOTE: If the molecular index is introduced in the First Strand Synthesis Primer, a paired-end sequencing run is required for read-out.
Partial Illumina P5 Adapter Sequence (Read 1) for Second Strand Synthesis Primer:
5' CACGACGCTCTTCCGATCT – NNNNNN(NN) – Target sequence (mRNA-sequence) 3'Anchor
UMIs to control sequencing run quality
Another advantage of including a random sequence or UMI is that it will avoid cluster registration errors on Illumina platforms. Illumina instruments rely on the presence of all four nucleotides ( A, C, G, and T) at each position of the initial sequencing cycles, to ensure accurate cluster registration. If using only one, or a few targeted primers the sequence diversity of the library at the start of one or both sequencing reads can be very low (or zero if only 1 target-specific primer is used). Low diversity sequences present at the start of Illumina sequencing reads (cycles 1 - 5) can thus result in cluster loss and reduced run outputs.
If random nucleotides / UMIs are not included in your primers, be sure to design and combine your targeted primers in such a way that all four nucleotides will be represented in each of the first 5 cycles of both sequencing reads.