View Single Post
Old 03-07-2017, 09:01 AM   #1
SDPA_Pet
Senior Member
 
Location: US

Join Date: Apr 2013
Posts: 222
Default Illumina HiSeq library insert size

Hello, I did a illumina HiSeq 2X150 bp metagenomic sequencing recently. I have some questions.

1>I have got my sequencing report back (from sequencing center). The report says the average insert size is about 600bp, which means majority of reads that was prepare to be sequenced are around 600bp. I am confused about it. You know, after I got my fastq files back (R1 and R2). I firstly merged paired ends. I have >60% of reads that can be join together successfully. I don't how could this happen. Since the method only sequence 150 bp, and the fragment is 600 bp. There will be no overlaps (150 X 2 = 300 bp << 600 bp). Why I can still get so many reads joined. Let says, if I want to join more paired - end reads, the fragment size should be designed less than 300 bp right?

2> The report also says "300 cycles using the HiSeq system". This straight-forward. I suppose for R1 and R2 is 150 cycles, receptively. Each cycle will add one nucleotide and 150 cycle will be 150 bp. The sequencing center says they can also do maximum 500 cycles, which means 2X250 bp sequencing. I was wondering why they don't run more cycles such as 1000 cycles, so we could get 2X500 bp. This will give us longer reads. I don't know which factors restrict the illumina reads lengths? For the reports, it seems we can increase cycles to get longer reads.

Thanks,
SDPA_Pet is offline   Reply With Quote