I have some FASTQ files in two datasets which are sequences from 16Srna region. The first dataset is amplicons form V4 region and the second is V3-V4 region.

However all the reads are 250 nucleotides long, whereas one region is strictly included in the other. So what is the biological meaning of the length?

I'm expecting reads to have the same length as the sequenced/amplified region. I don't know the size of the regions but one is obviously longer than the other.

Thank you (I thought it was better to ask here rather than on bioinformatics.stackexchange.com)

  • 2
    $\begingroup$ This is absolutely on topic here, I just wanted to let you know that it would also be on topic on Bioinformatics as decided in this meta thread. Which of the two sites you choose is completely up to you. $\endgroup$ – terdon Aug 9 '17 at 13:03
  • $\begingroup$ Could edit your question and clarify what seems strange to you? What length of reads were you expecting? What you describe is standard but something seems to be confusing you. What is that? $\endgroup$ – terdon Aug 9 '17 at 13:04
  • $\begingroup$ I edited my question after @terdon comment. $\endgroup$ – abichat Aug 9 '17 at 13:10

The read length has absolutely nothing to do with what you are sequencing. It is a characteristic of the sequencing technology you use. NGS sequencing techniques typically produce this sort of short read you're seeing. The read length does not change because you are sequencing a longer molecule. You would still get ~250nt reads even if you were sequencing an entire genome. Your reads are something like this (image source):

image showing reads aligned to a genome

So the vast majority of your 250nt are overlapping and cover slightly different parts of your target sequence. This is one of the reasons why NGS analysis is not trivial. The first step in any NGS analysis is to assemble your reads into a bam file that covers your target region. If you need help on doing that, come on over to http://bioinformatics.stackexchange.com.

  • $\begingroup$ Thanks you for you answer. But there is something I don't understand: with this technique of "cutting the molecule" that has to be sequencing, where do you put your barcodes? (when there are multiple samples from different sources) $\endgroup$ – abichat Aug 9 '17 at 14:11
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    $\begingroup$ @MrSnake on the end of the read but that really should be a separate question. $\endgroup$ – terdon Aug 9 '17 at 14:20

My understanding is that if the reads come straight from the sequencing machine, they will all be the same length. That corresponds to the number of sequencing cycles that the machine was set to perform. This has no biological meaning.

I don't know what the machine will read once it reads more than the length of the fragment subjected to sequencing.

If the fragments are shorter than what the sequencer reads, there will be some library preparation adapters to remove from the sequences in order to recover the actual fragments. Then you should be able to see the real fragment lengths.

If the fragments are longer than what the sequencer reads, see @terdon's answer.


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