Optimal Insert Size for TruSeq V2 Sequencing

Dario1984 · 04-04-2019, 09:59 PM

Illumina's TruSeq Sample Preparation Guide contains a table showing how to modify RNA fragmentation time to get different median insert lengths using the divalent cations and heat method in Appendix B. The median length ranges from 140 to 200 bases. Aren't all of these options wasteful if paired-end 150 bases sequencing is done? I think the ideal inset size would be 300 bases in that case, but if 0 minutes is done, the table shows 200 bases as the median size and you can't do less than 0 minutes! Is there an alternative method which doesn't result is mainly reverse-complemented pairs of reads? Our collaborators' sequencing service uses 8 minutes fragmentation and a plot of the insert sizes shows the mode of the distribution is about 120 bases and the median is about 140 bases so, the majority of reads not only are completely reverse complements of each other, but also have much (or all) of the TruSeq adapter at their ends. The reads are each 150 bases long.

nucacidhunter · 04-05-2019, 02:56 AM

Generally smaller inserts would result in more uniform coverage of genes so for counting applications short inserts are preferred and can be sequenced only 70 cycles. For other applications such as transcriptome assembly larger fragments and high number of sequencing cycles are more efficient. Some kits such as NEBNext Ultra II enable better customisation of insert size ranging from 150bp-1kb.

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04-04-2019, 09:59 PM	#1
Dario1984 Senior Member Location: Sydney, Australia Join Date: Jun 2011 Posts: 166	Optimal Insert Size for TruSeq V2 Sequencing Illumina's TruSeq Sample Preparation Guide contains a table showing how to modify RNA fragmentation time to get different median insert lengths using the divalent cations and heat method in Appendix B. The median length ranges from 140 to 200 bases. Aren't all of these options wasteful if paired-end 150 bases sequencing is done? I think the ideal inset size would be 300 bases in that case, but if 0 minutes is done, the table shows 200 bases as the median size and you can't do less than 0 minutes! Is there an alternative method which doesn't result is mainly reverse-complemented pairs of reads? Our collaborators' sequencing service uses 8 minutes fragmentation and a plot of the insert sizes shows the mode of the distribution is about 120 bases and the median is about 140 bases so, the majority of reads not only are completely reverse complements of each other, but also have much (or all) of the TruSeq adapter at their ends. The reads are each 150 bases long.

04-05-2019, 02:56 AM	#2
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Generally smaller inserts would result in more uniform coverage of genes so for counting applications short inserts are preferred and can be sequenced only 70 cycles. For other applications such as transcriptome assembly larger fragments and high number of sequencing cycles are more efficient. Some kits such as NEBNext Ultra II enable better customisation of insert size ranging from 150bp-1kb.