Multiple lane reads achieved from Illumina NovaSeq 6000 for downstream analysis

Question

[a.bibek52]

Heya!

I used Illumina NovaSeq6000 for the genome sequencing using SP lane, 2*250 PE reads, and obtained the sequencing results as:

SNP21357007_S1_L001_R1_001.fastq
SNP21357007_S1_L001_R2_001.fastq
SNP21357007_S1_L002_R1_001.fastq
SNP21357007_S1_L002_R2_001.fastq

Now, I want to perform the downstream analysis of the result that I obtained, however, I am confused about whether I have to use the PE reads independently for each used lane and consider one as a technical replicate or I need to merge the single reads obtained from two lanes together for the downstream processing.

I am not being able to find a valid answer from the literature as they do not illustrate how they used the multiple-lane results. Therefore, if anybody was stuck on a similar problem earlier or is familiar with this kind of issue, can you please help me with the valid documentation (if available)?

Thank you.

Answer 1

[GenoMax]

You can merge the lane specific files together before doing any processing. People find it useful to process them in parallel until a point (creating aligned BAMs) and then merge the BAM's for final processing. This allows parallelization and speed up of processing.

Here is some info on how you can merge the files: Concatenating fastq.gz files across lanes

[a.bibek52]

Thanks GenoMax for your quick reply. Do you have any documentation that mentions about merging the files across lanes together for downstream analysis?

[GenoMax]

Lanes on Ilumina flowcells are not always physically separate even though they may be optically so. The same pool runs on multiple lanes of some NovaSeq flowcells unless a XP kit is used that allows addressing individual lanes when one can put distinct samples/pools on the lanes. It is possible to use a parameter during initial data processing to generate single files for each sample.

It is the same pool of samples running across all lanes of a FC so the data can be merged together for analysis.

See: https://knowledge.illumina.com/software/cloud-software/software-cloud-software-reference_material-list/how-to-concatenate-the-fastq-files-from-different-lanes

[a.bibek52]

Thank you for the help, GenoMax.