Hi guys,
I am a newbie for Illumina sequencing. I just got my sequencing results back from a sequencing center. I am doing environmental microbial amplicon sequencing (16S rRNA). The center uses paired-end. Here is what I think. When they do paried-end sequencing. They sequences twice. First, use forward primer to sequence (5'--3'). Second, use reverse primer to sequence from other direction (3'-5'). For each sample, I have to sequence results and I can assemble them to a large sequences (contig, whatever it called).
Here are files that I got from the sequencing center
1> They give me several files: fasta, qual, fastaq files. These three files are same origin. I was told they are RAW data that have been joined (for amplicons > 300 and < 570bp). Also, I was told that they are joined reads as well as reorienting the 3’-5’ reads into a uniform direction.
Here is my first question: When I check the joined reads. I only find forward primers and barcodes, but there are no reverse primers and barcodes. Does this mean they have removed the reversed primers+barcodes. I suspect they only did a single direction sequencing. My reads is kind of short. The median is ~ 150bp after remove primers and barcodes
2> They also gave me two text files. According to them, if you amplicons are short (<300bp), they don't join them. Theoretically, my amplicons are longer than 300bp, based on the length between the forward and reverse primers. I don't know why they still give me these two files.
Inside the text file, it looks somthing like this
M02542:80:000000000-AFKAB:1:1116:23915:4775
M02542:80:000000000-AFKAB:1:1115:7131:7895
Does illumina sequencing generate txt file? I just switch from 454 to illumina sequencing. Text file in 454 sequencing is raw flowgram file. What does text file in illlumina mean. I haven't heard illumina generates txt file before.
3>Last, I need to redesign some of my old 454 primers for Miseq sequencing. Do you know the maximum length of amplicon I can make for paired-ends sequencing in Miseq
I am a newbie for Illumina sequencing. I just got my sequencing results back from a sequencing center. I am doing environmental microbial amplicon sequencing (16S rRNA). The center uses paired-end. Here is what I think. When they do paried-end sequencing. They sequences twice. First, use forward primer to sequence (5'--3'). Second, use reverse primer to sequence from other direction (3'-5'). For each sample, I have to sequence results and I can assemble them to a large sequences (contig, whatever it called).
Here are files that I got from the sequencing center
1> They give me several files: fasta, qual, fastaq files. These three files are same origin. I was told they are RAW data that have been joined (for amplicons > 300 and < 570bp). Also, I was told that they are joined reads as well as reorienting the 3’-5’ reads into a uniform direction.
Here is my first question: When I check the joined reads. I only find forward primers and barcodes, but there are no reverse primers and barcodes. Does this mean they have removed the reversed primers+barcodes. I suspect they only did a single direction sequencing. My reads is kind of short. The median is ~ 150bp after remove primers and barcodes
2> They also gave me two text files. According to them, if you amplicons are short (<300bp), they don't join them. Theoretically, my amplicons are longer than 300bp, based on the length between the forward and reverse primers. I don't know why they still give me these two files.
Inside the text file, it looks somthing like this
M02542:80:000000000-AFKAB:1:1116:23915:4775
M02542:80:000000000-AFKAB:1:1115:7131:7895
Does illumina sequencing generate txt file? I just switch from 454 to illumina sequencing. Text file in 454 sequencing is raw flowgram file. What does text file in illlumina mean. I haven't heard illumina generates txt file before.
3>Last, I need to redesign some of my old 454 primers for Miseq sequencing. Do you know the maximum length of amplicon I can make for paired-ends sequencing in Miseq
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