Dear all, My Version of QIIME 2 is qiime2-2020.2 installed in conda. I have two fastq files PairEnded 2X250 bp. Sequences are demultiplexed because barcodes are not contained in the sequence but were in the header. [Captura de Pantalla 2020-04-21 a la(s) 10.16.38 a. m.] I have imported sequenc…

Hi @victoriamesa , This looks like a problem with your core sequence file. If they're available, I would start by contacting your sequencing provider and letting them know that you have sequences where quality scores don't match the length of the sequence. This is a red flag that there may have bee…

**Hi @jwdebelius ** I really appreciate your help. Thank you so much. **I have contacted the sequencing provider but I don't have answer yet. ** During this I I've been able to import sequence in manifest format with the following qzv result: qiime tools import ** --type 'SampleData[PairedEndSe…

Hi @victoriamesa , [image] victoriamesa: Mismatched forward and reverse sequence files: 78208, 77691 This means that you've got a different number of sequences in your forward and reverse file. It again points to problems with your file that you should discuss with your sequencing provider. T…

Hi @jwdebelius I have the original files without demultiplexing. Here the files: [R1] [R2] I have successfully imported them with the following code: qiime tools import --type MultiplexedPairedEndBarcodeInSequence --input-path MULTIPLEXED/ --output-path MULTIPLEXED/multiplexed-seqs.qza I…

Hi @victoriamesa , Personally, I would try both and see what gives the best results, especially if this is your first time with this protocol/machine/provider. I will just say it's hard to determine what exactly is correct for your specific data/protocol/provider because everyone has something that'…

Hi @jwdebelius , thank you for your previous reply. I would like you to review the procedure that I have developed. I have some doubts about the results. qiime cutadapt demux-paired \ --i-seqs multiplexed-seqs.qza \ --m-forward-barcodes-file metadata.tsv \ --m-forward-barcodes-column Barcode \ --p-…

Hi @victoriamesa , You may want to consider your quality filtering parameters; it looks like you're losing a lot of sequences there. You might want to trim the sequences shorter, although you generally have low quality reads. Best, Justine.

Thank you @jwdebelius , What kind of parameters could you recommend me?

Hi @victoriamesa , You need to look at your data, and consider what will work for you. There is no one correct answer. You want to a balance sequence length and sequence quality. I would consider where you see large drop-offs in quality and use that to trim. Best, Justine

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