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.
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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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Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...-
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Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...-
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